Colchester ended Gillingham's unbeaten record after running out 2-1 winners in an entertaining match at the Weston Homes Community Stadium.
Cody McDonald's shot struck a post inside two minutes for Gills before Colchester took a fourth-minute lead through Gavin Massey, who finished clinically from Callum Harriott's pass.
But Gillingham levelled three minutes later when Luke Norris ran on to McDonald's flick and slotted smartly low past Jamie Jones.
Jones denied Josh Wright before Harriott restored Colchester's lead just before the half hour with a sweet left-foot strike from Matthew Briggs' short pass.
Gillingham were awarded a penalty shortly afterwards when Frankie Kent was ruled to have handled Brennan Dickenson's cross in the area but Jones smartly saved McDonald's spot-kick.
Jones then made a superb double save just after half-time to deny Jordan Houghton and then McDonald, while Bradley Dack sent a header straight at the U's goalkeeper.
Jones foiled Dack late on and Max Ehmer went close for Gillingham but they could not prevent a first defeat of the season.