The pruritogenic potency of tryptase and its involvement in anti-pruritic effect of intravenous nafamostat mesilate (NFM) were studied in mice. An intradermal injection of tryptase (0.05–1 ng/site) elicited scratching in ICR mice, while chymase was without effects at doses of 0.05–50 ng/site. The dose–response curve of tryptase action was bell-shaped and the effect peaked at 0.1 ng/site (≈0.7 fmol/site). NFM (10 mg/kg) inhibited scratching induced by tryptase but not by histamine and serotonin. NFM (1–10 mg/kg) produced the dose-dependent inhibition of scratching induced by intradermal compound 48/80 (10 μg/site). The inhibition by NFM (10 mg/kg) was abolished in mast cell-deficient (WBB6F1 W/W^V) mice, but not in wild-type (WBB6F1 +/+) mice. NFM (10 mg/kg) suppressed tryptase activity in the mouse skin. Proteinase-activated receptor-2 (PAR-2) neutralizing antibody (0.1 and 1 μg/site) and the PAR-2 antagonist FSLLRY (10 and 100 μg/site) inhibited scratching induced by tryptase (0.1 ng/site) and compound 48/80 (10 μg/site). These results suggest that mast cell tryptase elicits itch through PAR-2 receptor and that NFM inhibits itch-associated responses mainly through the inhibition of mast cell tryptase.