Rovers have improved drastically in League One after a tricky start to life back in the third tier, whereas Colchester will be looking to distract themselves from a League Two relegation battle with a successful run in this competition.
Match preview
The EFL Trophy has been a welcome escape for Colchester from their League Two worries, with Matt Bloomfield's men topping Southern Group A ahead of League One's Charlton Athletic after going unbeaten throughout the three-game group stage.
A victory over The Addicks, success against Brighton & Hove Albion's Academy and a share of the spoils with fellow fourth-tier strugglers Gillingham secured The U's a home tie in the second round with the aim now to see how far they can go in this year's competition.
Colchester have competed in the EFL Trophy ever since their relegation from the Championship 14 years ago, but have only made it past this stage on one occasion when they reached the semi-finals in the 2008-09 season before losing to Luton Town at Kenilworth Road.
In fact this is only the second time in six years that Colchester have progressed past the group stage, so it is fair to say long-time supporters of the club will not be expecting a miraculous cup run and a Wembley appearance despite being unbeaten in the competition so far.
The home faithful at the JobServe Community Stadium would much rather their side retain their league status this season but a victory over Rovers on Wednesday night would improve the mood around the club nevertheless.
Bristol Rovers were pipped to top spot in Southern Group E due to a penalty shootout loss to Plymouth Argyle in the first match of the group stage but still come into this game as one of the seven sides in this section of the competition that are unbeaten in normal time.
After dramatic scenes on the final day of last season which secured Rovers automatic promotion to League One, The Pirates have settled nicely on their return to the third tier after a concerning start which saw them win just twice in 11 matches.
Central to that upturn in form has been the prolific antics of Welshman Aaron Collins, who has netted 10 goals in 19 matches so far and only trails Peterborough United's Jonson Clarke-Harris in the league's scoring charts.
Before a disappointing group-stage exit last campaign, Bristol Rovers had reached at least the third round in the EFL Trophy in the previous three seasons and will be confident in getting back to the latter stages of the competition against a Colchester side who are suffering at the bottom end of League Two.
Colchester United EFL Trophy form:
W
W
L
Colchester United form (all competitions):
D
D
L
L
L
W
Bristol Rovers EFL Trophy form:
L
W
W
Bristol Rovers form (all competitions):
D
D
L
W
D
W
Team News
Colchester boss Bloomfield is set to name a strong XI as he looks to cause an upset on Wednesday night and is likely to include his key players in the action given that The U's next game after this clash is a week on Saturday.
Eighteen-year-old forward Samson Tovide scored his first goal of the season in the 3-0 win over Doncaster Rovers on the weekend and should be rewarded with another start up front here, with the academy graduate a player the Rovers defence will need to keep a keen eye on.
Ex-Celtic man Scott Sinclair scored the decisive goal for Bristol Rovers in the win over Peterborough on Saturday and Joey Barton will want to get the most of the 33-year-old, who could be recalled by Swansea City and loaned elsewhere when the January transfer window opens in a few weeks.
Unlike their hosts, The Pirates are likely to change their starting XI considerably from the weekend with a key second-round FA Cup tie against Boreham Wood to come on Sunday, meaning the likes of Luke McCormick and John Marquis could be brought into the side.
Colchester United possible starting lineup:
Hornby; Tchamadeu, Chambers, Smith, Kazeem; Judge, Ashley, Newby, Hannant, Nouble, Tovide
Bristol Rovers possible starting lineup:
Belshaw; Connolly, Thomas, Gibson; Sinclair, Rossiter, Finley, Gordon, McCormick; Coburn, Marquis
We say: Colchester United 0-1 Bristol Rovers
Both sides come into this knockout tie after important home wins in their respective leagues at the weekend, so neither will be fearing their opposition and should be eyeing an enjoyable run in the competition.