Colchester's 100 per cent record came to an end thanks to goals from star striker Tcham N'Toya-Zoa.
The 21-year-old Congolese forward hit a superb strike after 15 minutes, and although Colchester pulled level with a goal from Gavin Johnson on the stroke of half time, the visitors never looked like taking anything from the game.
Ten mintues after the break N'Toya-Zoa put Chesterfield ahead again after sweeping home Ian Evatt's header.
Stephen Hunt marked his Colchester debut by being sent off within 60 seconds of coming on as a 65th minute substitute.
His first act was to cruelly chop down N'Toya-Zoa near the halfway line and after referee Andy Hall had sorted out a 20-man scuffle he showed debutant Hunt the red card.
Although Colchester pushed Chesterfield back despite being down to 10 men, it was the home side who squandered a host of chances to put the result beyond doubt.
N'Toya-Zoa twice spurned opportunities for his hat-trick, while Caleb Folan also missed when it seemed odds on he would score.