Full-time from Florence Park
Millwall's proud unbeaten run came to an abrupt halt as a first-half brace from Colchester United's Macauley Bonne inflicted a first defeat in 10 games on the young Lions.
In a quiet opening half an hour at Florence Park neither side registered a shot on target until the 32nd-minute, when a spectacular fingertip save by U's keeper James Bransgrove kept out a sumptuous curling effort from Alfie Pavey.
The hosts broke the deadlock on 39 minutes when Bonne stabbed home from close range after keeper Tom King could only parry his initial effort.
Colchester made it 2-0 with their second just four minutes later when Bonne capitalised on some hesitancy in the visiting defence and fired home his second of the game.
Lions keeper King was called into action five minutes after the break, producing a splendid save to keep out Dion Sembie-Ferris' goalbound effort.
Millwall had King to thank again in the 58th-minute as he denied Sembie-Ferris and then Kane Vincent-Young in quick succession.
Despite enjoying a fair amount of possession thereafter, the young Lions were unable to create any clear scoring opportunities, ending any hope of extending that proud unbeaten run to 10 games.
Afterwards coach Scott Fitzgerald commented: "It was disappointing to see our recent run come to an end in this way. On a bobbly pitch we made two mistakes and they cost us dearly.
However, we put a very young side out there today including three first year scholars and I couldn't fault any of them for effort and application.
We now have to make sure we round the Development League season off in positive fashion on Friday at Sheffield Wednesday."
Colchester United: Bransgrove, Vincent-Young, Olufemi, James, Harney, Jenkins, Sembie-Ferris Curtis, 65mins), Lawrence, Bonne, Bailey (Marriott, 65mins), Dunne. Subs not used: Boness, Stephens, Curtis, Marriott, Sheriff.
Millwall: King, Siafa, Casey, Webb (Fitzgerald, 76mins), Parr, Brown, Eze, Chesmain, Pavey, Philpot, Ndjoli (Mundle-Smith, 64mins). Subs not used: Barton, Mulrooney-Skinner.