The present study was undertaken to evaluate antidiabetic and antioxidant activities of Cayratia trifolia root extract against streptozotocin induced diabetes in experimental rats to scientifically validate its use against diabetes in some parts of India. Ethanolic extract, showing the highest activity in in vitro experiments, was prepared in saline and administered orally to streptozotocin induced albino Wistar diabetic rats for 21 days. Biochemical parameters liver and muscles glycogen and in vivo antioxidant activity in normal, diabetic control, standard (metformin) and treated animals were determined and compared. Attempt was made to isolate, purify and characterize one of the major secondary metabolites in extract by range of chromatographic and spectroscopic techniques. Treatment of streptozotocin induced diabetic rats with ethanolic root extract (500 mg/kg) caused significant (P < 0.01) reduction in blood glucose (312-178 mg/dL), increase in body weight (181-219 g) and serum insulin (1.28-2.26 IU/dL). It also maintained lipid profile and tests of liver and kidney functions within normal range as compared to diabetic control rats and almost at par with standard drug metformin. The oxidative stress induced decline in glutathione and catalase in liver and kidney tissues showed recovery nearly to normal level as a function of treatment. The GC-MS profile of the extract showed relatively high concentration of beta-sitosterol which was characterized by different spectroscopic and chromatographic techniques. The result scientifically and comprehensively validate the reported use of roots of this indigenous plant against diabetes. A strong antioxidant activity of the ethanolic root extract suitably compliments the antidiabetic effect.