A second half brace from the league's top scorer earn Cambridge United all three points against the U's, with Michael Folivi on target for Steve Ball's side from the penalty spot.
Steve Ball's Colchester United side lined up at the Abbey Stadium with two changes from the weekend's draw against Port Vale.
Jevani Brown and Noah Chilvers were dropped to the bench, with Courtney Senior and Luke Gambin coming into the starting eleven.
There was an early chance for the home side with Paul Mullin trying his luck with fifteen seconds on the clock. The league's top scorer looked to curl one when he cut inside, but Gerken had the goal covered and was able to punch away.
The U's looked dangerous in the opening ten minutes as well, with Harriott and Senior proving a real threat down the wings. Harriott went close to opening the scoring after ten minutes when he danced through the home defence, beating Iredale but he could only blast his shot over the bar from a tight angle.
Cambridge United went closest yet to finding the net, albeit unintentionally. Jack Iredale's cross flicked off the top of Gerken's crossbar and out for a goal kick with nineteen minutes gone.
Callum Burton in the home goal was tested for the first time in the game with twenty-five minutes played as he palmed away an effort from Norris. Luke's swerving effort seemed to catch out the home 'keeper who was diving to his right but had to change direction and push the effort away.
Burton was just an audience member for the next U's shot, when Poku cut in to the box, but the young winger couldn't quite find the target with the ball just missing the far post.
Poku had another run ten minutes later that would have been a brilliant goal had he been able to apply the finish. He took it through three Cambridge defenders, creating the space and cutting into the box, but he blasted the ball over the bar, to the amusement of the home fans behind the goal.
The hosts had the best two chances before the half time whistle blew though, and both chances came just as the game entered first half injury time, with Dean Gerken pulling off two fine saves.
The first was a free kick from Ipswich Town loanee, Idris El Mizouni. He got the ball up and over the wall, but couldn't beat Gerken who went flying across his goal to push the ball out for a corner.
And from the resulting corner, the visiting 'keeper made his best save of the half. Harry Darling got his head to the ball, rising highest in between the U's defence and sent his header towards the far corner with Gerken somehow clawing the ball off the line and out to safety.
Cambridge United started the second half the same as they had ended the first, but this time they got their noses in front. The league's top scorer Paul Mullin showed why he has scored so many goals this season, stooping low to nod the ball into the corner of the net from a Hannant free kick.
Steve Ball quickly made a triple change to his side, as he did at Port Vale to try and get the U's back in to the game, but Mullin added a second ten minutes after the first.
Knibbs got away from the U's defence down the right flank when his cross/shot found Mullin unmarked at the back post, with an easy tap in to double his tally and his side's lead.
But the two goal advantage didn't last long when Colchester United grabbed one back with just over twenty minutes to play. Folivi was tripped in the area, and stepped up to take the penalty himself. He sent Burton the wrong way, making the score 2-1.
The U's kept pressing and looked dangerous going forwards with Noah Chilvers and Michael Folivi both having shots on goal, but without seriously testing Burton.
Jevani Brown's quickly taken free kick seemed to free Harriott down the right wing, but the U's winger could only find the side netting with time running out at the Abbey Stadium.
Cambridge took their opportunity with the game coming to a close to make changes to, suring up their defence, with the U's unable to break through at the back and snatch an equaliser.
The home side took all three points, with the U's tasting defeat for the first time in three games. Steve Ball's side remain in the play-off positions, sitting in seventh, ahead of their next opponents, Morecambe on goal difference.
Cambridge United starting XI: Burton, Knoyle, Iredale, Digby, Taylor, Darling, Hannant, Mullin (Ironside 90'), Cundy, El Mizouni (Davies 89'), Knibbs.
Unused subs: Mitov, Dallas, May, Worman, Neal.
Colchester United starting XI: Gerken, Welch-Hayes (Tchamadeu 71'), Bramall, Smith, Senior (Chilvers 59'), Norris (Brown 59'), Harriott, Eastman, Poku, Stevenson, Gambin (Folivi 59').
Unused subs: George, Sowunmi, Clampin.