The striker was handed his first start of the season at Southend on Boxing Day ahead of Kevin Lisbie, who is due back at parent club Ipswich in the new year.
Gillespie, who has been plagued by hamstring trouble since joining the U’s 18 months ago, pulled up in the second half at Roots Hall and was taken off as a precaution to be replaced by Lisbie.
Another forward, Kayode Odejayi, misses out following the end of his loan spell from Barnsley but he will seal a permanent switch to the Community Stadium on January 1 and will be available against Preston in the FA Cup next week.
Scott Vernon (knee), Lee Beevers (shoulder) and Ashley Vincent (ankle) are still sidelined.
Meanwhile, manager Aidy Boothroyd praised his side for keeping their cool after they secured a 2-1 victory over local rivals Southend at Roots Hall.
Second-half goals from Phil Ifil and Anthony Wordsworth put the U’s in control but Lee Barnard pulled one back to set up a grandstand finish.
However, Colchester held on to secure a fifth win in six games, much to the delight of Boothroyd.
He said: “On a big occasion like a derby it is easy to get caught up in it but we did not do that. We had a game plan and we stuck to it and it worked.
“It was a tough game. We knew they are a good team and they are a handful. They have some good players and we stopped them playing and imposed ourselves.
“We were the better team in the first half and in the second half they came out all guns blazing but we managed to weather that.”