AbstractListeria monocytogenesis a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine againstListeriais available. AttenuatedListeriastrains offer protection and are tested as antitumor vaccine vectors, but would benefit from a better knowledge on immunodominant vector antigens. To identify novel antigens, we screen forListeriapeptides presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detect 68Listeriaimmunopeptides from 42 different bacterial proteins, including several known antigens. Peptides presented on different cell lines are often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations results in specific CD8+ T-cell responses and induces protection in vaccination challenge experiments in mice. Our results can serve as a starting point for the development of a clinical mRNA vaccine againstListeriaand aid to improve attenuatedListeriavaccines and vectors, demonstrating the power of immunopeptidomics for next-generation bacterial vaccine development.