Updated project metadata. Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8 T-cell responses, but very few T cell epitopes are known in mice as most common animal infection model for listeriosis. To identify epitopes we screened for Listeria epitopes presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. In between more than 6,000 mouse self-peptides presented on MHC Class I molecules, we detected 26 Listeria peptides from 25 different bacterial proteins, including previously reported antigens. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to CD8 T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. Despite its high biological potency in a prime boost model, this epitope did not protect against challenge infection but can be used as a research tool to probe CD8 T cell responses in mouse models of Listeria infection. Our results demonstrate the power of immunopeptidomics for bacterial antigen identification but highlight the need for in-depth immune characterization for vaccine candidate selection.