Both sides boast four points from their opening two matches in Southern section Group E, where Leyton Orient and MK Dons are hoping to upset the apple cart in the final round of fixtures.
Match preview
Still waiting for their first slice of success in the EFL Trophy - making it to the final in 1996-97 before being defeated by Carlisle United - Colchester's quest for a place in this year's last 32 got off on the right foot with an opening 2-1 success over MK Dons.
However, Danny Cowley's men missed an opportunity to top the rankings on matchday two, where they left it late to draw level with Leyton Orient but fell short in the bonus-point penalty shootout, where they only converted two of their four attempts.
As a result, the League Two unit currently find themselves in the runners-up spot courtesy of an inferior goals scored tally to upcoming visitors Arsenal, but their fate is in their own hands; win and they top the group, draw and win the penalty shootout and they top the group, draw and lose the penalty shootout and they should go through anyway.
Third-placed MK Dons and last-placed Leyton Orient are both two points worse off than Arsenal's Under-21s and Colchester, but the latter are severely lacking inspiration at present, as a 1-1 draw at Barrow on Saturday stretched their winless run in all tournaments to nine games.
That sequence includes a 2-1 FA Cup first-round loss at the hands of Swindon Town on November 2, and their nine-year stay in the EFL is under serious threat; they lie 21st in the League Two rankings, only three points off the bottom.
Similarly, Arsenal's Under-21s were left to rue a missed opportunity on matchday two of the EFL Trophy, having secured their own 2-1 victory away to Leyton Orient on September 3 before a stellar fightback against MK Dons was for nought.
Twice Mehmet Ali's team fell behind, and twice they drew level against the League Two side to force penalties, but they fluffed their lines and then some from the 12-yard mark, missing each of their first three attempts in the shootout and going home without a bonus point.
Nevertheless, the Gunners' are currently sitting pretty at the summit of Group E and are also safe in the knowledge that they are in control of their knockout destiny, but there has not been much for the visitors to shout about in recent weeks.
Indeed, Arsenal have been beaten in four of their last five games in all tournaments - most recently suffering a 2-1 loss to Chelsea in the Premier League 2 - and Ali's team have conceded at least twice in all of those matches, letting in an alarming 15 goals in total.
Even in their only victory in that sequence, Arsenal shipped three to Blackburn Rovers before managing to scrape a 4-3 victory, and their underwhelming run of form has bumped them down to sixth place in the Premier League 2 table, six points off Manchester City at the summit.
Colchester United EFL Trophy form:
W
L
Colchester United form (all competitions):
L
L
D
L
L
D
Arsenal Under-21s EFL Trophy form:
W
L
Arsenal Under-21s form (all competitions):
W
L
L
L
W
L
Team News
In the EFL Trophy, players incur a one-game suspension for picking up just two yellow cards; as Colchester defender Rob Hunt has been cautioned in both of his side's games so far, he is banned for the visit of Arsenal.
Mandela Egbo or Oscar Thorn should fill in for Hunt on the right-hand side here, and the hosts have also lost three players to international call-ups - Lyle Taylor to Montserrat and the duo of Aaron Donnelly and Jamie McDonnell to Northern Ireland's Under-21s.
There is one familiar name to Gooners in Colchester's ranks, goalkeeper Matt Macey - who was on the books at the Emirates from 2013 to 2021 - but the 30-year-old has not earned any minutes in the EFL Trophy this season and is not expected to start against his former youth team here.
Meanwhile, Arsenal stand to lose five players to international duty this month, including goalkeeping trio Alexei Rojas, Tommy Setford and Lucas Nygaard, Albanian defender Maldini Kacurri and first-team fringe player Myles Lewis-Skelly.
The latter was never expected to be a part of the squad for Tuesday's game anyway, and with Nygaard due to link up with the Denmark squad, Brian Okonkwo could be given the nod in between the posts.
Further forward, Nathan Butler-Oyedeji - who has made Premier League and Champions League squads under Mikel Arteta in the current campaign - should captain the Gunners at the tip of the attack.
Colchester United possible starting lineup:
Smith; Thorn, Egbo, Goodliffe, Iandolo; Read, Woodyard; Anderson, Hopper, Gordon; Tovide
Arsenal Under-21s possible starting lineup:
Okonkwo; Robinson, Monlouis, Heaven, Nichols; Rosiak, Gower, S. Oulad M'Hand; Kabia, Butler-Oyedeji, Edwards
We say: Colchester United 2-2 Arsenal Under-21s (Colchester win on penalties)
Tuesday's tie has all the makings of a goal-laden thriller, as Arsenal's defending has been nothing short of woeful over the past few weeks, but Colchester are set to be working with limited options at the back.