Aminopeptidases that catalyze the removal of N-terminal residues from polypeptides or proteins are crucial for physiological processes. Here, we explore the biological functions of an M29 family aminopeptidase II from Listeria monocytogenes (LmAmpII). We show that LmAmpII contains a conserved catalytic motif (EEHYHD) that is essential for its enzymatic activity and LmAmpII has a substrate preference for arginine and leucine. Studies on biological roles indicate that LmAmpII is required for in vitro growth in a chemically defined medium for optimal growth of L. monocytogenes but is not required for bacterial intracellular infection in epithelial cells and macrophages, as well as cell-to-cell spreading in fibroblasts. Moreover, LmAmpII is found as dispensable for bacterial pathogenicity in mice. Taken together, we conclude that LmAmpII, an M29 family aminopeptidase, can efficiently hydrolyze a wide range of substrates and is required for in vitro bacterial growth, which lays a foundation for in-depth investigations of aminopeptidases as potential targets to defend Listeria infection.