Craig Westcarr gave Chesterfield an early lead, but a brace from Jordan Robertson and goals from Josh Walker and Andy Barcham turned the game on its head.
Scunthorpe were protecting a seven-game unbeaten record, but Chesterfield made the early running and went in front in the 21st minute when Westcarr rode two challenges to beat visiting goalkeeper Sam Slocombe from 10 yards.
But the home side's lead lasted only eight minutes before Conor McAleny, on loan from Everton, crossed from the left and Robertson smashed a shot into the roof of the net.
Scunthorpe went in front in the 65th minute when a neat exchange of passes ended with McAleny pulling the ball back for Walker to sweep a shot inside Chesterfield goalkeeper Tommy Lee's left-hand post from 10 yards.
The game was over 10 minutes later when Robertson curled a 20-yard shot into the top corner and Barcham completed an emphatic victory when he carved through the defence to roll a shot past Lee.
Source: DSG