Isoleucine, a branched chain amino acid, plays an important role in the improvement of glucose metabolism as evidenced by the increase of insulin-independent glucose uptake in vitro. This study evaluated the effect of isoleucine on glucose uptake and oxidation in fasted rats, and gluconeogenesis in vivo and in vitro. Oral administration of isoleucine decreased the plasma glucose level by 20% and significantly increased muscle glucose uptake by 71% without significant elevation of the plasma insulin level when compared to controls at 60 min after the administration. Furthermore, expiratory excretion of 14CO2 from [U-14C]-glucose in isoleucine administered rats was increased by 19% as compared to controls. Meanwhile, isoleucine decreased AMP levels in the liver, but did not affect hepatic glycogen synthesis. Under insulin-free conditions, isoleucine significantly inhibited glucose production when alanine was used as a glucogenic substrate in isolated hepatocytes. This inhibition by isoleucine was also associated with a decline in mRNA levels for phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), and a decreased activity of G6Pase in isolated hepatocytes. These findings suggest that a reduction of gluconeogenesis in liver, along with an increase of glucose uptake in the muscle is also involved in the hypoglycemic effect of isoleucine. In conclusion, isoleucine administration stimulates both glucose uptake in the muscle and whole body glucose oxidation, in addition to depressing gluconeogenesis in the liver, thereby leading to the hypoglycemic effect in rats.