One of the major side effects of anti-cancer chemotherapy is mucosal injury in the gastrointestinal tracts. 5-Hydroxytryptamine (5-HT) is critically involved in intestinal injury due to its role as a paracrine messenger, neurotransmitter, and inflammatory mediator. Nafamostat mesylate (nafamostat), a potent and specific serine protease inhibitor, has been reported to have a therapeutic effect on several types of gastrointestinal inflammation including experimental colitis. In this study, we investigated whether nafamostat ameliorates methotrexate-induced gastrointestinal injury and/or potentiation of 5-HT dynamics in intestine. Rats received methotrexate intraperitoneally a consecutive administration (12.5 mg/kg/day) for 4 days. Nafamostat (1 or 3 mg/kg) was given subcutaneously 10 min before first administration of methotrexate, and then every 24 h for three consecutive days. Jejunal tissues were collected to analyze. Nafamostat 1 mg/kg, but not 3 mg/kg, ameliorated methotrexate-induced villus atrophy, as well as mRNA expression of pro-inflammatory cytokines. Also, nafamostat 1 mg/kg significantly inhibited methotrexate-induced tryptophan hydroxylase1 mRNA expression and 5-HT contents. The present study suggests that nafamostat could be applied as a therapeutic agent for gastrointestinal injuries caused by anti-cancer drugs.