Objective To provide experimental method and technical support for the establishment of a rhesus monkey model of human Coxsackie B2 virus infection for analyzing the infection characteristics of human Coxsackie B2 virus in rhesus monkey models. Methods Coxsackie B2 virus was inoculated orally to infect infant rhesus monkeys aged 3-4 months. The clinical symptoms, body weight and temperature of the rhesus monkeys were observed. Blood was collected to detect physiological and biochemical indexes. Virus titers were analyzed in the blood and herpes tissues. Herpes tissues were pathologically examined. Results Herpes appeared on the hands, feet and mouth 2-5 days after infection; animals showed depression, decreased activity and diet intake. Coxsackie B2 virus was detected in the herpes and serum samples. Rhesus monkeys infected with the virus routinely developed leukopenia, and had increased numbers of monocytes, granulocytes, and eosinophils, but decreased numbers of lymphocytes and basophils. Blood biochemical tests showed that the liver function, kidney function, and myocardial enzymes were more or less increased. Herpes histopathology indicated that the tissues had squamous epithelial thickening, excessive surface hyperkeratosis, local necrosis with inflammatory cell infiltration, and abscess formation. Conclusions Characteristic effects of viral infection in infant monkeys including hand, foot and mouth herpes, clinical symptoms, physiological and biochemical impairments, and viremia, indicating that the rhesus monkeys were successfully infected with Coxsackie B2 virus orally. The established detection method and techniques used were feasible for this type of study and laid an experimental foundation for the subsequent establishment of a model of rhesus Coxsackie B2 virus infection for pathogenesis research and drug and vaccine evaluation.