Bristol City took the lead through a Scott Murray strike on two minutes but Colchester struck back as a Gareth Williams strike and a brace from Neil Danns clinched the game for them.
Marcus Stewart pulled a goal back on 90 minutes but Colchester now sit pretty in second place.
Meanwhile, towards the top of League One Brentford played out a 1-1 home draw with play-off chasing Nottingham Forest.
Kris Commons's deflected free-kick put the visitors in front early before DJ Campbell equalised after 34 minutes.
Forest then had new signing Julian Bennett sent off for a second bookable offence, whilst Bees manager Martin Allen was sent to the stands in the final minutes.