Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor and is expressed throughout the gut. It is well known that PAR-2 participates in the regulation of gastrointestinal motility; however, the results are inconsistent. The present study investigated the effect and mechanism of PAR-2 activation on murine small intestinal smooth muscle function in vitro. Both trypsin and PAR-2 activating peptide SLIGRL induced a small relaxation, followed by a concentration-dependent contraction. The sensitivity to trypsin was greater than that to SLIGRL (EC₅₀=0.03 vs 40µM), but maximal responses were similar (12.3±1.6 vs 13.7±1.3 N/cm2). Trypsin-evoked contraction (1µM) exhibited a rapid desensitization, whereas the desensitization of response to SLIGRL was less even at high concentration (50µM). Atropine had no effect on PAR-2 agonist-induced contractions. In contrast, TTX and capsaicin significantly attenuated those contractions, implicating a neurogenic mechanism that may involve capsaicin-sensitive sensory nerves. Furthermore, contractions induced by trypsin and SLIGRL were reduced by NK-1 antagonist, SR140333, or NK-2 antagonist, SR48968, alone, or further reduced by combined application of SR140333 and SR48968, indicating the involvement of neurokinin receptors. In addition, desensitizing neurokinin receptors with substance P and /or neurokinin A decreased the PAR-2 agonist-evoked contraction. We concluded that PAR-2 agonists induced a contraction of murine intestinal smooth muscle that was mediated by nerves. The excitatory effect is also dependent on sensory neural pathways and requires both NK1 and NK2 receptors.