Hoffenheim, meanwhile, are aiming to reach the quarter-finals of the competition for the first time in eight seasons.
Match preview
Leipzig were staring a third defeat in the DFB-Pokal final in four seasons in the face when Marcel Halstenberg saw red for a professional foul with his side already trailing Freiburg 1-0 last campaign.
However, Christopher Nkunku's late equaliser forced their opponents to penalties, with Leipzig finally lifting the club's first ever major trophy after several near misses in recent years.
Having hugely improved under Marco Rose in recent months, after their cup-winning manager Domenico Tedesco was sacked in September after making a miserable start to the season, the former Borussia Dortmund boss will sense a great opportunity to win his first trophy as a manager in German football.
His side still remain in the Champions League, of course, but with Pep Guardiola's Manchester City awaiting them in the last-16, their chances of progressing in that competition are extremely slim.
Given the patchy form of Bayern Munich, perhaps a more likely prospect for Leipzig is winning their first ever Bundesliga title after moving two points behind the Bavarians with a 2-1 win against Stuttgart on Friday night.
In the meantime, though, Rose will be determined to progress beyond Hoffenheim on Wednesday and move into the last eight of the DFB-Pokal, with Die Roten Bullen capable of becoming one of the few teams in German history to retain the competition.
Despite being bank-rolled to greater success in a similar fashion to Leipzig in recent years, Hoffenheim have a miserable record in Germany's primary domestic cup.
Die Kraichgauer have failed to progress beyond the last-16 of the DFB-Pokal in each of the last seven seasons, having fallen at the quarter-final stage in six of the previous eight campaigns.
The club have never gone beyond the last eight in this competition, so Andre Breitenreiter could create a new chapter in Hoffenheim's history should he be able to take them deep into the latter stages of the cup.
Based on current form, though, springing an upset away to Leipzig on Wednesday does not appear to be a likely proposition. Their 4-1 defeat at home to Borussia Monchengladbach last time out was their fifth loss in their last six league games, having only beaten rock-bottom Schalke 04 within their last 12 games in the Bundesliga.
However, perhaps a change in competition will provide a welcome distraction for Breitenreiter and his players, even if the former FC Zurich's boss' position in charge must be increasingly insecure.
RB Leipzig DFB-Pokal form:
W
W
RB Leipzig form (all competitions):
W
W
W
D
W
W
Hoffenheim DFB-Pokal form:
W
W
Hoffenheim form (all competitions):
L
L
L
L
D
L
Team News
Dani Olmo was subbed at half-time of his side's win against Stuttgart due to a hamstring injury, which could keep the Spain midfielder sidelined until the end of February.
Abdou Diallo, Peter Gulasci and Nkunku all remain unavailable, with Rose likely to provide the likes of Yussuf Poulsen, Emil Forsberg and Konrad Laimer starts after they came off the bench on Friday.
Hoffenheim, meanwhile, will travel to Leipzig without Angelo Stiller, Kevin Vogt, Robert Skov, Kevin Akpoguma, Jacob Bruun Larsen and Grischa Promel due to injury.
Ihlas Bebou will almost certainly return to the starting XI after being dropped without any positive impact on Saturday, with Kasper Dolberg likely to make way.
RB Leipzig possible starting lineup:
Blaswich; Klostermann, Simakan, Halstenberg, Raum; Laimer, Haidara; Szoboszlai, Forsberg; Poulsen, Werner
Hoffenheim possible starting lineup:
Baumann; Kabak, Brooks, Nsoki; Kaderabek, Rudy, Bischof, Angelino; Baumgartner, Kramaric; Bebou
We say: RB Leipzig 3-1 Hoffenheim
Although the hosts may make a few more changes to their side then their opponents, Leipzig's defence of their DFB-Pokal title should continue beyond Wednesday evening.
Hoffenheim arrive in eastern Germany on a horrible run of form, which could see their manager lose his job sooner or later if it does not improve soon.