Background: Rotavirus infection is the most common cause of infectious diarrhea and gastroenteritis among children worldwide. The viral proteins (VP), especially VP4- and VP7-induced neutralizing antibodies, were considered to be critical in protective immunity to rotavirus disease. However, whether the antibody to rotavirus nonstructural protein 4 (NSP4) protects against rotavirus-induced diarrhea directly is not completely clear, especially for the protective time course. Materials and Methods: To obtain direct evidence, 12-day-old ICR mice were treated with NSP4 and entire rotavirus to induce diarrhea. Results: Both NSP4 and rotavirus-treated mice developed diarrhea, which was accompanied by histological changes in the small intestine compared to age-matched control mice. Anti-NSP4 antibody demonstrated protection against both entire rotavirus-induced diarrhea and NSP4-induced diarrhea. The histological changes in the small intestinal were reversible. These data show that early intervention with anti-NSP4 antibody can prevent rotavirus-induced diarrhea in mice: late intervention with anti-NSP4 antibody could halt diarrhea progression in mice. Conclusions: Our findings demonstrate for the first time that administration of anti-NSP4 antibody is effective both prior to and during the time course of rotavirus infection. These observations extend our knowledge of rotavirus infection and its therapeutic options.