The Iron were edged out of the Capital One Cup losing 5-4 on penalties to Championship side Barnsley.
No goals were scored in 120 minutes of play which meant that spot kicks would determine the outcome of the match and Tykes striker Chris O’Grady kept his nerve to hand David Flitcroft’s team a place in the second round.
United made two changes to the side that faced Mansfield in the League Two curtain raiser three days previously with Christian Ribeiro replacing the suspended Eddie Nolan at right back and Sean McAllister coming in for Andy Welsh to make his competitive debut.
The home side made one change to the eleven that started their 4-0 defeat against Wigan at the weekend as Jim O’Brien came in for Scott Wiseman and following a cagey opening period, it was the hosts who had the game’s first opportunity after six minutes.
Striker O’Grady collected a long ball forward and cut inside onto his right foot before hitting a powerful effort towards the Iron goal that Sam Slocombe did well to save down low.
Jacob Mellis then also did well to create an opportunity for himself just outside the area but fired wide before Barnsley winger Jim O’Brien danced past a couple of United defenders and fired a low ball into the box that David Mirfin cleared under pressure.
Scunthorpe’s first chance came ten minutes into the match when Deon Burton robbed Tom Kennedy of the ball, 25 yards from goal and caressed a curling left footed effort that went just over the crossbar.
Andy Dawson later came close to breaking the deadlock as his free kick went inches past the Tykes upright after Terry Hawkridge had been fouled just outside the area.
The Iron then had two more opportunities in the space of a few seconds as first Matt Sparrow had an effort blocked before Hawkridge suffered a similar fate following an initial teasing cross from Ribeiro as United began to seize the initiative.
But the South Yorkshire side then forced Slocombe into further action as midfielder David Perkins collected Stephen Dawson’s pass and unleashed a forceful attempt that United’s no.1 did well to tip over after a deflection off Sparrow.
The chances dried up just before the break but O’Grady did have a snapshot at goal that fizzed past the post and Jean-Yves M’voto glanced a header wide from Tom Kennedy’s corner as the clocked ticked down on first half proceedings.
One minute was added on to the end of the opening period but 0-0 was how the game remained going into the interval.
It was Scunthorpe that produced the second half’s first chance as Sam Winnall won the ball midway through the Barnsley half and fed Hawkridge but he could only blaze his effort over when deep inside the 18 yard box.
Dawson went close when he wriggled past a cluster of opposition defenders before striking wide and Burton also tried his luck just inside the area to no avail as United had the better of the opportunities in the opening stages of second period.
Barnsley then had their first shot of the half as former Iron forward Chris Dagnall did well to control a lofted ball into the area and struck a powerful attempt at goal that went just over the crossbar.
Home shot stopper Luke Steele did well to save Winnall’s low drive as the chances began to come thick and fast at both ends of the pitch and just a few minutes later, O’Grady played Dagnall in on goal but his attempt flew just past Slocombe’s left hand post.
Brian Laws then made a double substitution as Andy Welsh and Chris Iwelumo both entered the fray and Scunthorpe remained resilient as the tie went into the last ten minutes, blocking numerous attempts at goal from the Championship club.
Dagnall almost grabbed a late winner for Barnsley at the end of normal time as he came within inches of connecting with O’Brien’s centre and Perkins also went close with a long distance free kick.
But goalless is how the game stayed after 90 minutes meaning that an extra half an hour would be played in order to try and decide a winner.
Tykes substitute Jason Scotland had the first chance of the opening period of extra time but could only hit over when well-placed and Slocombe also did well to save a Dagnall attempt on goal as Scunthorpe struggled to create any real goal scoring opportunities.
Scotland then won the ball in the air and saw his headed attempt trickle wide before Steele tipped a long distance Sparrow shot over the bar in the Iron’s only effort of the first 15 minutes.
Barnsley were again on top in the second period of extra time and had the best chance of the night, 112 minutes into the game.
O’Brien hooked a deep ball into the area back across goal but O’Grady’s volleyed attempt sailed well over the bar when unmarked.
Matt Godden then fired over for the Iron from deep inside the six yard box and Tomasz Cywka struck a fierce long distance free kick that Slocombe did well to tip away for a corner.
Godden was again played in on goal just seconds later but saw his effort saved by Steele and the end of extra time was signalled by referee Paul Tierney meaning that a penalty shootout would determine which team progressed to the second round.
Terry Hawkridge was first up for the Iron but hit his powerful attempt high over the bar and then Chris Dagnall did likewise for Barnsley before Michael Collins calmly slotted his penalty into the bottom right hand corner to give United the lead.
Jason Scotland missed again for the Tykes and then Matt Sparrow saw his attempt saved by Luke Steele before Tomasz Cywka netted to level things up.
Chris Iwelumo scored to give Scunthorpe the lead again and Stephen Dawson, Matt Godden and David Perkins all then converted their kicks.
Andy Welsh’s low left footed shot was saved before both goalkeepers blazed their efforts over the crossbar and then Jim O’Brien hit the crossbar.
David Mirfin scored to make it 4-3 and then Scott Golbourne levelled things up once again.
Niall Canavan’s spot kick was then saved by Steele which gave Chris O’Grady the chance to win it for David Flitcroft’s side.
He dispatched his penalty meaning that Barnsley won the shootout 5-4 and progressed to the second stage of the competition.
Next up for Scunthorpe is an away trip to Bristol Rovers in the league on Saturday.
Barnsley: Steele (c), Golbourne, Cranie (Wiseman, 63), M’voto, Kennedy (Cywka, 71), O’Brien, Perkins, Dawson, Mellis (Scotland, 54), O’Grady, Dagnall
Substitutes not used: Alnwick, Hassell, McNulty, Cofie.
Scunthorpe United: Slocombe, Ribeiro, Canavan, Mirfin, Dawson, Hawkridge, McAllister (Godden, 79), Collins (c), Sparrow, Winnall (Welsh, 71), Burton (Iwelumo, 71)
Substitutes not used: Severn, Waterfall, Howe, Hornsey
Attendance: 5,238 (496 away).