Rovers have improved drastically in League One after a tough start to life back in the third tier, whereas Colchester will be looking to distract themselves from a League Two relegation battle with a strong run in this competition.
The competition has been a welcomed escape from Colchester's fourth-tier worries, as they topped the Southern A group ahead of Charlton Athletic, going unbeaten in the three-game group stage.
Victories over both the Addicks and the Brighton academy were followed up with a share of the spoils with fellow League Two strugglers Gillingham to secure the U's a home tie in this, the second round.
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.
Colchester boss Matt Bloomfield is likely to name a strong 11 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.
18-year-old forward Samson Tovide scored his first goal of the season in a 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.
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: Hornby (GK), Tchamadeu, Chambers, Smith, Kazeem, Judge, Ashley, Newby, Hannant, Nouble, Tovide.
Bristol Rovers: Belshaw, Connolly, Thomas, Gibson, Sinclair, Rossiter, Finley, Gordon, McCormick, Coburn, Marquis.
As mentioned previously, the 2004-born Hackney boy scored his first senior goal in The U's previous fixture, and the highly-rated youngster will be keen to kick on against stern opposition in this one.
Where is the game being played?
The match will be played at the JobServe Community Stadium in Colchester.
What time is kick-off?
The game will kick off at 19:30 GMT.
How to watch
The game will unfortunately be unavailable to watch on live television, but game passes can be purchased to watch online via the club websites.