Menopause is associated with obesity and insulin resistance. However, the effects of estrogen on insulin sensitivity in ovariectomized rats fed on high-fat diet (HFD) remains unclear. In this study, we examined in vivo effects of estrogen on energy intake, body weight, fat accumulation, insulin sensitivity and insulin signaling in ovariectomized (OVX) rats fed on HFD or standard chow diet (SD). Female Wistar rats aged 9 week were ovariectomized. After 4 week, the rats were assigned either to a placebo-treated (Placebo; n=26) group or a group treated with 17β-estradiol (Estrogen; n=26) subcutaneously implanted with either placebo- or 17β-estradiol (1.5 mg/60-day release) pellets. Simultaneously, rats in either group were divided into two groups and given either SD (13% of calories from fat) or HFD (60% of calories from fat). Four weeks after the replacement and HFD treatment, intravenous glucose tolerance test (IGTT, 1g/kg) was performed for assessment of insulin sensitivity. Blood samples were taken at 0, 5, 10, 30 and 60 min from the catheter implanted into the right jugular vein of rat. Plasma glucose and insulin were measured by the glucose oxidase method using a glucose assay kit and by ELISA assay kit, respectively. Two days after the IGTT, 10-5 mol/l insulin was injected into the portal vein in each group of rats, and the liver, hind-limb muscle and mesenteric adipose tissue were dissected for insulin signaling analysis. The Akt and phosphorylated Akt in these organs were assayed by Western blotting. In the Placebo group, HFD increased energy intake compared with SD for 3 weeks after the diet started, while in the Estrogen group it decreased energy intake remarkably for 2 weeks. Four weeks after replacement and HFD, the accumulation of intra-abdominal fats was enhanced in the Placebo group, but was suppressed in either diet group of the estrogen-treated rats. The basal levels of plasma glucose and insulin concentrations were higher in the Placebo group than in the Estrogen group. IGTT revealed that insulin sensitivity was decreased in the Placebo group than that in the Estrogen group of rats fed on SD. Furthermore, HFD reduced insulin sensitivity in the Placebo group, but had little effects on the sensitivity in the Estrogen group. In addition, insulin injection increased Akt phosphorylation in the liver of the Estrogen group, but not in the Placebo group. These results suggest that HFD induces insulin resistance in the OVX rat by impairing insulin-stimulated Akt activations in insulin target organs, which are ameliorated by estrogen replacement.