Bristol City's 1-0 reverse at Millwall - thanks to a Paul Robinson strike after 78 minutes - mean the Robins now cannot catch the Iron.
Scunthorpe's defeat at Bloomfield Road did have one memorable moment though - as Billy Sharp's consolation was his 32nd goal of the season, setting a new club scoring record.
City's defeat means they face a nervous final day of the season to find out whether they will hold on to the final automatic promotion spot.
Nottingham Forest now trail them by just one point, with their 3-1 win at Leyton Orient keeping their promotion hopes alive.
Jack Lester hit a brace, with in-form Kris Commons also on the scoresheet. Matt Lockwood grabbed Orient's consolation.
Blackpool also have the chance of claiming second position, if both City and Forest slip-up.
Chesterfield thrashed Bradford 3-0 at Valley Parade, with both sides relegated at the final whistle after Cheltenham picked up all the points at Rotherham.
Yeovil's 1-0 win over Doncaster confirmed their play-off spot, while Oldham just held on to the final place in the end-of-season lottery thanks to a 2-1 success at Brighton.
Swansea, who meet Blackpool next week, are now the only team who can deny Oldham a play-off spot. The Swans kept up their promotion push with a 2-1 victory at Carlisle.
Crewe and Tranmere drew 1-1, as did Bournemouth and Gillingham.
Brentford's relegation was already confirmed, but they sealed a dramatic 4-3 win over Port Vale at Griffin Park.