Oral inoculation of human rotavirus MO strain (serotype 3) into 5-day-old BALB/c mice caused gastroenteritis characterized by diarrhea (90% on the average, on day 2). Using this animal model, preventive effect of antiviral agents on the development of rotavirus-induced diarrhea was examined. The infectivity of human rotavirus was enhanced by treatment with protease in vitro. A cysteine protease inhibitor, E-64-c, was given orally at 12hr and 24hr after MO infection. Oral administration of 0.3mg of E-64-c decreased the diarrhea ratio to 17.5% on day 2 and to 10% on day 3. Oral administration of 0.15mg of cysteine protease inhibitor, ovocystatin, completely prevented the diarrhea on day 2. Serine protease inhibitor, aprotinin (0.15mg×2), also prevented the diarrhea on day 2 to 14.3%. These protease inhibitors were nontoxic in vitro and to suckling mice. The histopathological changes in the small intestine due to infection recovered 2 days after MO infection in mice treated with E-64-c and ovocystatin. These results suggest that protease inhibitors are protective agents for human rotavirus infection by inhibiting proteases required for viral replication.