Dental pulp inflammation often results from the dissemination of periodontitis caused mostly by Porphyromonas gingivalis infection. Calcitonin gene-relatedpeptide (CGRP) and Substance P (SP) are proinflammatory neuropeptides that increase in injured pulp and hypersensitive teeth. Recently, we demonstrated that a purified trypsin-like proteinase, a gingipain from P. gingivalis called RgpB, induced the release of neuropeptides from human pulp cells (HPC) mediated by protease-activated receptor (PAR)-2 activation. Nafamostat mesilate (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate; NM), a potent serine protease inhibitor that is now in clinical use, was found to inhibit enzyme activities of human tryptase. Objectives: We explore the effects of NM on RgpB-induced CGRP and SP release and on the inducible pathway by which NM affects the release. Methods: NM's effect on RgpB activity was measured by using a specific substrate (Boc-Phe-Ser-Arg-MCA). The effect of NM on RgpB-induced neuropeptides release was investigated using RT-PCR and ELISA. PAR-2-like immunoreactivity (PAR-2-LI) and neuropeptides-LI were localized on human dental pulp by immunohistochemistry and immunofluorescence assay. The activations of mitogen-activated protein kinases (MAPKs) and ATF-2 were examined by Western blot analysis. Results: NM suppressed the proteolytic activity of RgpB in a dose-dependent manner with an IC50 of 1.0x10-7 M. NM showed no effect on the release of RgpB-induced neuropeptides after the PAR-2 gene was knocked out from HPC. This indicated that NM's inhibitory effect was dependent on the PAR-2 signaling pathway. Neither NM nor these oligonucleotides exerts a cytotoxic effect on HPC when determined by MTT assay. An immunofluorescence assay indicated the downregulation of both neuropeptides-LI following NM treatment. The kinase inhibitor profile showed that NM blocked p38 MAPK and p44/42 downstream of PAR-2 activation, and subsequently attenuated the release of neuropeptides in HPC. Conclusion: The activity of NM, a new trypsin-like inhibitor, suggests a possible candidate drug for pulpitis accompanied by neuropeptides-associated dental pain and hypersensitivity.