A stoppage-time strike from Will Randall broke U's hearts as Sutton United twice came from behind to snatch all three points in South London.
Colchester United lead twice in the match with Owura Edwards netting three minutes into his debut, before Sutton equalised six minutes later through an Isaac Olaofe strike.
But Freddie Sears was on hand to convert his twelfth goal of the season from the penalty spot after Louis John had handled the ball in the eighteen-yard area.
The home side turned it around in the second half though, with Olaofe bagging his second of the game before a late curling strike from Will Randall gave the home side all three points.
Hayden Mullins named a line up with four changes to the last eleven, that started at home to Barrow on Saturday afternoon.
Tommy Smith, Luke Hannant, Owura Edwards and Emyr Huws were all given starts, with the latter their first for the club - replacing Alan Judge, Corie Andrews, Junior Tchamadeu and Cameron Coxe.
Owura Edwards made a fantastic start to his Colchester United career with a goal inside the opening three minutes. The latest addition to the U's squad worked with Brendan Wiredu to pick up the ball, drive towards goal and shoot, with Edwards' effort wrong-footing Bouzanis and flying into the bottom corner.
But the hosts were quick to level the score in the game, making it 1-1 after just nine minutes. It was an unfortunate goal for the U's to concede, with Bugiel's initial effort saved onto the woodwork by Turner, with Isaac Olaofe tapping in the rebound.
The hosts were close to turning it around with a quarter of an hour played, but Jake Turner was up to the challenge. The U's on-loan 'keeper got across well to push away Omar Bugiel's looping header that looked destined for the top corner, denying Sutton United the lead.
Through an even first half, it was Jake Turner who was worked a little more, but it was the U's that were leading at the break, courtesy of a Freddie Sears penalty. Luke Hannant worked hard to fashion a shot from a tight angle, with Bouzanis parrying the ball to Sears at the back post, who saw his effort deflect off the arm of Louis John.
The referee pointed to the penalty spot, and Freddie duly dispatched his twelfth of the season to make it 2-1 to Hayden Mullins' side with the teams heading for the dressing rooms and a fifteen minute break.
After half time, the U's were able to restrict the hosts to only efforts from outside of the area, with Turner not really tested by Sutton's attackers.
Freddie Sears had a half-chance to make it 3-1 to Colchester United, when Louis John's header was too light to make it back to Dean Bouzanis. Sears nipped in to take the ball, but poked his effort wide, with the angle closed down and his path blocked by the home 'keeper.
And with half an hour to play, the hosts managed to force home an equaliser after a mix up in the U's penalty area. It was Isaac Olaofe who managed to get the equaliser, and his second of the game heading home after Jake Turner had managed to palm away an initial effort.
And Jake Turner was called upon once again ten minutes before the full-time whistle after Bennett had fired a cross in at pace. Ricky Korboa was close to finding the net after the ball had squirmed out of Turner's hands, but Cole Skuse was able to clear the lines for the visitors.
But it wasn't until the nintieth minute when the home side took the lead in the match, at a decisive time. Will Randall collected the ball on the left flank, cutting inside Welch-Hayes and striking a low drive into the bottom corner to make it 3-2.
The U's remain in twenty-second place, with twenty-two points. Up next is another away game, with Hayden Mullins' side taking on Salford City on Saturday afternoon.
Sutton United starting XI: Bouzanis, Goodliffe, John, Bugiel (Bennett 63'), Randall, Eastmond, Olaofe (Wilson 76'), Boldewijn (Korboa 74'), Kizzi, Milsom, Smith.
Unused subs: Barden, Wyatt, Davis, Nelson.
Colchester United starting XI: Turner, Welch-Hayes, Chambers, Smith (Eastman 90'), Wiredu, Hannant, Skuse, Sears, Edwards (Nouble 72'), Dallison, Huws.
Unused subs: Gerken, Judge, Chilvers, Andrews, Coxe.