United were defeated 3-2 away at Sheffield Wednesday in League One on Saturday.
The Iron went behind in the opening five minutes as Gary Madine tapped home.
The visitors had a penalty given and then over-ruled before Ben Marshall thumped home the Owls’ second on 17 minutes.
Bobby Grant scored middway through the second half to make a game of it, but Madine soon added his second and Wednesday’s third.
Mark Duffy made an impact coming on as a substitute however, pulling one back and coming close to levelling the scores on two other occasions.
The Iron entered the match making no changes from the team beaten 2-1 after 120 minutes in the Carling Cup second round against Newcastle United in midweek.
Josh Lillis started in goal, with Andrew Wright, Michael Nelson, Niall Canavan and Eddie Nolan forming the defence.
The midfield consisted of Jimmy Ryan, Oliver Norwood, Sam Togwell and Andy Barcham, while Chris Dagnall and Bobby Grant led the attack.
The substitutes were Sam Slocombe, Garry Thompson, Paul Reid, Mark Duffy and Michael Collins.
United had the first chance of the game in the second minute when a long ball was only cleared as far as Grant, who half-volleyed a spectacular effort over the crossbar.
The Owls went ahead in the fourth minute however as Madine forced home from close range after Nolan had been beaten by Marshall down the right flank and the winger crossed for the former Millwall striker, who tapped in from four yards.
Almost immediately after, Scunthorpe were handed a penalty as James Linington pointed to the spot when Dagnall turned David Prutton and appeared to be felled just inside the area.
However, the referee changed his mind after speaking to his linesman and the penalty was changed into a corner, with Togwell’s low effort on the rebound being cleared away to safety.
The Owls doubled their advantage on 17 minutes when Marshall charged down the left side of the area and cut inside before firing a rasping drive towards goal which rose above and flew past Lillis.
Scunthorpe looked to find a way back into the game and a goal almost came on the half-hour mark when Dagnall was played through down the left by Ryan’s excellent throughball. His shot across goal beat the goalkeeper but clipped the post.
The first chance of the second period came the way of the Owls, for whom Marshall struck a fierce effort which was tipped over the bar by Lillis.
United were still looking for a way back into the game and had a half-chance when Dagnall’s run into the area was stopped and Norwood blazed the rebound over the crossbar.
Then, Barcham came close, seeing his volleyed effort from the left side of the area deflected wide for a corner.
The left-winger continued to be a threat and his deep cross from the left was headed wide of his own goal by Julian Bennett. Then, from the subsequent corner, Togwell volleyed an attempt wide.
On 63 minutes, Scunthorpe came even closer as Canavan’s header from Ryan’s right-wing corner delivery landed on the roof of the net
Scunthorpe made their first substitution a short while afterwards as Thompson came on for Ryan, and then Collins replaced Togwell..
And he made an instant impact, winning a header which played in Dagnall, who teed up his strike partner Grant to slot the ball beyond the onrushing Nicky Weaver from around 14 yards in the 67th minute.
The Owls made it three as Madine was played through by Jose Semedo down the right side of the area and struck the ball past Lillis, who got a touch but the ball trickled into the back of the net.
United instantly looked to force their way back into it, and Grant forced a close range block from Weaver after a scramble down the left side of the area.
The next Iron alteration saw Barcham replaced by Duffy and, soon after, Canavan headed wide from a corner as Scunthorpe continued to try and seek a second goal.
The Owls were always a huge threat on the break though and had a chance when Chris Lines played in Madine, who struck an effort just wide from the edge of the area.
Duffy pulled a consolation back on 85 minutes as a cross into the area was only part cleared and the winger smashed an effort into the back of the net from the left side of the area.
Two minutes later, the winger came within a whisker of levelling the scores but his header across goal was superbly tipped out by Weaver.
Then, in the first minute out of four in stoppage time, Duffy unleashed a thunderous right-footed strike from the edge of the area which went just wide of goal.
It proved Scunthorpe’s last chance, with the Iron returning to action on Tuesday night when they host Hartlepool United in the first round of the Johnstone’s Paint Trophy.
Attendance: 16,862 (550 away approx)
Report from Hillsborough