The worldwide rapid increase in diabetes poses a significant challenge to current therapeutic approaches. Single-dose mesenchymal stem cell (MSC) infusion ameliorates hyperglycemia but fails to restore normoglycemia in diabetic animals. We therefore hypothesized that multiple intravenous MSC infusions may reverse hyperglycemia in type 2 diabetes (T2D) rats. We administered serial allogenous bone-marrow derived MSC infusions (1 × 10(6)cells/infusion) via the tail vein once every 2 weeks to T2D rats, induced by high-fat diet and streptozocin (STZ) administration. Hyperglycemia decreased only transiently after a single infusion in early-phase (1 week) T2D rats, but approximated normal levels after at least three-time infusions. This normal blood level was maintained for at least 9 weeks. Serum concentrations of both insulin and C-peptide were dramatically increased after serial MSC infusions. Oral glucose tolerance tests revealed that glucose metabolism was significantly ameliorated. Immunofluorescence analysis of insulin/glucagon staining revealed the restoration of islet structure and number after multiple MSC treatments. When multiple-MSC treatment was initiated in late-phase (5 week) T2D rats, the results were slightly different. The results of this study suggested that a multiple-MSC infusion strategy offers a viable clinical option for T2D patients.