Many first-team players found themselves ruled out by injury, including Matthew Briggs, Kurtis Guthrie, Denny Johnstone, Frankie Kent, Lewis Kinsella, Doug Loft, Luke Prosser, Craig Slater, and Sammie Szmodics.

Darren Ambrose, Joe Edwards, Tosin Olufemi, Elliot Parish, Nicky Shorey and Marvin Sordell would all be leaving after the club decided to not renew their contracts following relegation to League Two.

[17] Colchester made their third and fourth summer signings on 28 June, bringing in Scottish striker Denny Johnstone from Birmingham City for an undisclosed fee,[18] and Courtney Senior from Brentford on a free transfer.

[20] Having spent the second half of the previous season on trial from Chelmsford City, defender Dexter Peter joined the club on a one-year contract on 29 June.

[26] Kurtis Guthrie became John McGreal's eighth summer signing on 6 July when he joined on a two-year contract from National League side Forest Green Rovers for an undisclosed fee.

[31] In their second pre-season friendly, Szmodics scored a hat-trick against Needham Market with Chris Porter converting from the penalty spot in a 4–1 win for Colchester on 12 July.

[34] Another Szmodics hat-trick and Macauley Bonne's second goal of pre-season proved to be the difference between Colchester and Chelmsford City as they earned another 4–1 win on 23 July.

[40] In the second week of the new league season, Colchester earned their first win of the campaign and John McGreal's first as manager as the U's won 2–0 at home against Cambridge United on 13 August.

Young left-back JJ Wilson joined Maldon & Tiptree on loan for one month,[43] while former Barnet trainee full-back Brendan Ocran signed a one-year development contract.

[51] The club made two transfer deadline day loan signings of winger Tarique Fosu from Reading until 7 January 2017,[52] and midfielder Glen Kamara for the same period.

[71] Callum Harrison also left the club temporarily on 25 October, joining Needham Market for a second loan spell with the Isthmian League Premier Division side.

[72] Also leaving the club on loan was Ben Wyatt, who joined National League South outfit Concord Rangers for one month on 28 October.

[73] Referee Kevin Johnson was forced to leave the field on a stretcher during Colchester's tie with Plymouth Argyle following a collision with Jimmy Spencer on 29 October.

Owen Garvan made his first appearance of the season, assisting a goal as Colchester earned their first victory in eleven League Two matches, moving them out of the bottom two.

[81] The same day, Dion Sembie-Ferris joined Ben Wyatt at National League South side Concord Rangers on loan for one month.

The U's took the lead after 15-minutes when Brennan Dickenson headed in a corner from Owen Garvan before Kurtis Guthrie added a second from the penalty spot after being fouled by former Colchester loanee Jamie Jones.

Josh Sheehan opened the scoring for Newport, before Chris Porter converted a penalty kick in the 35th minute after Kurtis Guthrie had been fouled in the area.

[95] Further transfer activity followed on 20 January, with triallist Tommy O'Sullivan joining Eli Phipps in moving from Cardiff City for an undisclosed fee.

[99] On 30 January, Chris Regis became the third Academy player to leave the club in order to seek first-team football,[100] while Rekeil Pyke joined the U's from Huddersfield Town on loan until the end of the season.

The arrival of goalkeeper Dean Brill following his release from Motherwell would provide competition for Sam Walker and would allow Dillon Barnes to head out on loan, in response to the surprise retirement of James Bransgrove in December.

Jamille Matt had opened the scoring for the home side after 12-minutes, but Kurtis Guthrie's tenth goal of the season gave Colchester a share of the points six minutes later.

A late consolation goal from Brennan Dickenson reduced the deficit, while both Charley Edge and Rekeil Pyke made their professional debuts from the bench as the club slipped to 10th in the league table.

First-half goals from Billy Kee and Matty Pearson handed Accrington the advantage before Omar Beckles was shown a red card for a tackle on Brennan Dickenson just before the break.

George Elokobi scored with a long range effort on 13-minutes, and the U's held on for the remainder of the game, while the visitors had defender Joe Jacobson sent off for a second bookable offence two minutes from full-time.

[113] Meanwhile, Femi Akinwande joined his third loan club of the season, signing with National League South East Thurrock United for one month on 3 March.

[124] Winger Tarique Fosu was named Man of the Match and earned a place in the EFL 'Team of the Week' for his performance after playing a part in two of Colchester's goals.

Two hours following the postponement, the players returned to the pitch in front of an empty stadium to play out the final five minutes plus three for stoppage time which sealed the 3–1 result for Colchester.

[130] Ahead of Colchester's final game of the season, it was revealed that Brennan Dickenson had sustained cruciate ligament damage during training that would leave him sidelined for up to nine months.

[132] McGreal named seven changes to his squad that drew 1–1 with Hartlepool on the opening day of the season for their EFL Cup tie with Championship side Brighton & Hove Albion, including debuts for young goalkeeper Dillon Barnes, Brennan Dickenson, and Doug Loft.

[133] In the new-look EFL Trophy group stage, Colchester lost their first game 1–0 to Crawley Town on 30 August, but handed first-team debuts for Dexter Peter and Diaz Wright.