Rotavirus (RV) infects pancreatic islets in vitro and is a potential trigger of type 1 diabetes. To study the pancreatic effects of RV in vivo murine RV (MRV) or rhesus RV (RRV) was administered to weanling mice. Tissue RV was quantified by ELISA and culture-isolation. Cell death and proliferation, and insulin expression, were analysed by immunohistochemistry. Following RV, two phases of transient hyperglycemia were observed beginning at 3 and 9 days. In the first phase, apoptosis of endocrine and exocrine cells was associated with a marked decrease in pancreas mass and insulin expression and secretion, but virus was not detected in the pancreas. These effects were mimicked by injection of poly I:C, a double-stranded RNA mimic, and were not observed in toll-like receptor 3 (TLR3)-deficient mice. By the second phase, the pancreas had regenerated but islets remained smaller and irregular and now contained virus. Thus, RV infection triggered acute pancreatic involution and hyperglycemia by a TLR3-dependent mechanism without directly infecting the pancreas; following rapid but incomplete recovery, a second phase of hyperglycemia was associated with pancreatic infection by RV. These findings support the view that RV may be an etiological agent in type 1 diabetes.