Serine proteases elicit cellular responses via protease-activated receptor-2 (PAR-2) which is known to regulate inflammation and the immune response. Although the gastrointestinal tract is exposed to large amounts of proteolytic enzymes, the role of PAR-2 in canine inflammatory bowel disease (IBD) remains unclear. The objective of this study was to investigate the effects of PAR-2 activation on inflammatory cytokine/chemokine gene expression in canine intestine and the expression of intestinal PAR-2 and fecal serine protease activity in dogs with IBD. Duodenal biopsies from healthy dogs were cultured and treated ex vivo with trypsin or PAR-2 agonist peptide, and inflammatory cytokine/chemokine gene expression in the tissues was then quantified by real-time PCR. PAR-2 mRNA and protein expression levels in the duodenal mucosa were examined by real-time PCR and immunohistochemistry, respectively. Fecal serine protease activity was determined by azocasein assay. In ex vivo-cultured duodenum, trypsin and PAR-2 agonist peptide induced significant up-regulation of mRNA expression levels of interleukin-1 β (IL-1β), IL-8, mucosae-associated epithelial chemokine (MEC) and fractalkine, and this up-regulation was inhibited by a serine protease inhibitor. Duodenal PAR-2 mRNA and protein expression levels were higher in dogs with IBD than in healthy control dogs. Fecal serine protease activity was significantly elevated in dogs with IBD, and the level of activity correlated positively with the clinical severity score. These results suggest that PAR-2 may contribute to the pathogenesis of canine IBD by inducing expression of inflammatory mediators in response to luminal serine proteases.