Citrobacter rodentium is a bacterial pathogen that causes a murine infectious colitis equivalent to enterohemorrhagic Escherichia coli infection in humans. Colonic luminal fluid from C. rodentium-infected mice, but not from sham-infected mice, contains active serine proteinases that can activate proteinase-activated receptor-2 (PAR₂). We have identified granzyme A and murine trypsins to be present in C. rodentium-infected luminal fluid, as determined by mass spectrometry and Western blot analysis. Inflammatory indices (colonic mucosa macroscopic damage score, increased intestinal wall thickness, granulocyte infiltration, and bacterial translocation from the colonic lumen to peritoneal organs) were all increased in C. rodentium-infected mice, compared with sham-infected mice. Soybean trypsin inhibitor-treated wild-type mice and untreated PAR₂-deficient (PAR₂^-/-) mice (compared with their wild-type littermates) both had substantially reduced levels of C. rodentium-induced inflammation. These data point to an important role for both pathogen-induced host serine proteinases and PAR₂ in the setting of infectious colitis.