Abstract: Objective To investigate the role of immunodeficiency and intestinal mixed infection on inducing extraintestinal dissemination of rotavirus (RV). Methods Immunodeficiency was induced in healthy Kunming mice by introperitoneal injection of cyclophosphamide, and RV was administered either orally or via intraperitoneal injection. In another group, toxigenic E.coli and human RV were given sequentially by intragastric administration to induce mixed infection. Three days later the organs of the mice were taken for pathological examination, and RV was detected by in situ PCR and hybridization. In children with or without viremia of rotavirus, blood tests for levels of tumor necrosis factor α (TNF-α), interleukin-2 (IL-2) and 7 trace elements (zinc, iron, copper, lead, calcium, manganese, and magnesium) were performed. Results In immunodeficient mice, pathological changes were found in the small intestinal villus, gastric lamina propria and the cardiac cells of mice taking RV orally, and the mice with intraperitoneal RV injection showed additional liver and kidney pathologies. In mice with mixed infections, pathological changes occurred in the intestines, livers and kidneys. In situ hybridization detected RV in the intestinal villius of immunodeficient mice with oral RV administration, and in the intestinal villus and kidneys of the mice with mixed infections. In situ PCR revealed the presence of RV in the intestinal villus, intestinal gland cells, epithelial cells of the proximal convoluted tubules and collecting tubes in the kidneys of immunodeficient mice taking RV orally, in the intestinal villus, kidneys, livers, hearts and pancreases of those with RV injection, and in the intestines, kidneys, and livers of the mice with mixed infection. Children with rotavirus viremia had TNF-α level in comparison with those free of rotavirus viremia, and the majority of the former children showed disorder in trace elements. Conclusion Immunodeficiency, mixed infection and malnutrition can be important factors contributing to or exacerbating RV infection and extraintestinal RV dissemination.