This study was intended to assess the effect of Gymnema sylvestre leaf extract on the AMPK-GLUT4 mediated signalling pathway on insulin resistance in polycystic ovarian syndrome-induced rats. For the induction of PCOS in rats, 1 mg/kg of letrozole was given orally for 21 days, after that metformin (300 mg/kg), Gymnema sylvestre leaf extract (200 mg/kg, and 400 mg/kg p. o.) were administered for further 28 days. Letrozole-induced PCOS rats illustrated significant estrus irregularity, and sex hormonal abnormality, and developed insulin resistance as indicated by increased fasting glucose levels, an increased rate of glucose clearance, and also decreases in GLUT4 and AMPK mRNA expression in ovarian cells. Ovarian histology in PCOS rats also showed many follicular cysts, atretic follicles, and the absence of the corpus luteum. These changes were significantly reversed by the treatment of G. sylvestre in a dose-dependent manner. It might be mediated through its insulin resistance modulating property by the activation of AMPK & GLUT4 expression on ovarian endometrial cells. © 2023 Elsevier B.V., All rights reserved.