Secreted proteins are one of the direct molecular mechanisms by which microbiota influence the host, thus constituting a promising field for drug discovery. Here, through bioinformatics-guided screening of the secretome of clinically established probiotics fromLactobacillus, we identify an uncharacterized secreted protein (named LPH here) that is shared by most of these probiotic strains (8/10) and demonstrate that it protects female mice from colitis in multiple models. Functional studies show that LPH is a bi-functional peptidoglycan hydrolase with both N-Acetyl-β-D-muramidase and DL-endopeptidase activities that can generate muramyl dipeptide (MDP), a NOD2 ligand. Different active site mutants of LPH in combination withNod2knockout female mice confirm that LPH exerts anti-colitis effects through MDP-NOD2 signaling. Furthermore, we validate that LPH can also exert protective effects on inflammation-associated colorectal cancer in female mice. Our study reports a probiotic enzyme that enhances NOD2 signaling in vivo in female mice and describes a molecular mechanism that may contribute to the effects of traditionalLactobacillusprobiotics.