Scunthorpe had their relegation to League Two confirmed, even though they beat sixth-placed Swindon 3-1.
All four goals came in the last 10 minutes, with Michael Collins, Michael Duffy and Karl Hawley giving the Iron the win after James Collins' opener.
They needed Colchester to lose to have any chance of staying up, but the Essex side won 2-0 at Carlisle, with goals from Gavin Massey and Tom Eastman securing their safety. Scunthorpe manager Brian Laws said: "It's a horrible taste, it's one that people will probably have thought about in the last couple of weeks in particular because it's not nice going back to that basement.
"Now that we know our destiny, we've got to regroup and move forward and that's certainly something the club as a whole needs to do along with the fans."