The purpose of this study was to evaluate the pancreatic beta cell protective and glucose uptake enhancing effect of the water extract of Tinospora cordifolia stem (TCSE) by using rat insulinoma (RIN)-m5F cells and 3 T3-L1 adipocytes. RIN-m5F cells were stimulated with interleukin-1β and interferon-γ, and the effect of TCSE on insulin secretion and cytokine-induced toxicity was measured by ELISA and MTT assay, respectively. The glucose uptake and protein expression were measured by fluorometry and western blotting. Antidiabetic effect of TCSE was measured using streptozotocin-induced diabetic rats. TCSE dose dependently increased cell viability and insulin secretion in RIN-m5F cells. In addition, TCSE increased both the glucose uptake and glucose transporter 4 translocation in 3 T3-L1 adipocytes via PI3K pathway.Finally, TCSE significantly lowered blood glucose and diet intake and increased body weight in streptozotocin-induced diabetic rats. The level of serum insulin and hepatic glycogen was increased, whereas the level of serum triglyceride, total cholesterol, dipeptidyl peptidase-4, and thiobarbituric acid reactive substances was decreased in TCSE-administered rats. TCSE also increased glucose transporter 4 protein expression in the adipose tissue and liver of TCSE-fed diabetic rats. Our results suggested that TCSE preserved RIN-m5F cells from cytokine-induced toxicity and enhanced glucose uptake in 3 T3-L1 adipocytes, which may regulate glucose metabolism in diabetic rats.