Michail Antonio's first-half strike was enough to secure Colchester a 1-0 win that sent Chesterfield crashing to their first home defeat since mid-August.
Leon Clarke struck a post for the home side, but Colchester defended well to claim their second away league win of the season.
Chesterfield started well with Alex Mendy and Clarke unsettling Colchester's defence, but the visitors worked their way into the game and went in front in the 30th minute.
A corner was only half cleared and when the ball broke to Antonio, he drove it low through a crowd of players and past Spireites goalkeeper Greg Fleming from 15 yards.
The home side almost equalised early in the second half when Mendy cut in from the right and beat two defenders, but visiting goalkeeper Ben Williams turned his shot around the post, while Anthony Wordsworth tested Fleming with a 25-yard shot and Clarke turned Nathan Smith's low cross on to the woodwork.
Chesterfield had strong claims for a penalty waved away when Clarke was challenged from behind and the striker saw a shot flash wide in stoppage time as Colchester held on.
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