ID1101 (4-hydroxyisoleucine), an amino acid extracted from fenugreek seeds, exhibits an interesting glucose-dependent insulin stimulating activity. The present study was undertaken to investigate a possible extrapancreatic effect of ID1101 on insulin signaling and action, besides its previously described insulinotropic action. Insulin sensitizing effects of ID1101 were investigated in rat in vivo by three different approaches, i) using euglycemic-hyperinsulinemic clamps in two different rat models of insulin resistance, i.e. rats fed a sucrose-lipid diet and Zucker fa/fa rats, ii) by measuring liver and muscle PI 3-kinase activity after an acute injection of ID1101 in normal and insulin resistant diabetic rats, and iii) after chronic treatment in two rat models of insulin resistance. Euglycemic hyperinsulinemic clamp experiments revealed that ID1101 is able to improve insulin resistance through an increase of peripheral glucose utilization rate in sucrose-lipid fed rats and by decreasing hepatic glucose production in Zucker fa/fa rats. Moreover, we demonstrated that a single injection of ID1101 activates the PI 3-kinase activity in liver and muscle from normal rats but also in muscle from diabetic rats. Finally, ID1101 chronic treatment significantly reduced insulinemia in type 2 diabetic rats and reduced the progression of hyperinsulinemia in insulin-resistant obese Zucker fa/fa rats. These findings clearly demonstrate that ID1101 is able to reduce insulin resistance through activation of the early steps of insulin signaling in peripheral tissues and in liver. In summary ID1101, besides its insulinotropic effect, directly improves insulin sensitivity, making ID1101 a potential very valuable therapeutic agent for diabetes treatment.