Abstract Postmenopausal women are at higher risk for obesity and insulin resistance due to the decline of estrogen, but genistein, a phytoestrogen, may reduce the risks of these diet-related diseases. In this study, we hypothesized that supplemental genistein has beneficial effects on insulin resistance in an ovariectomized rat model by modulating lipid metabolism. Three weeks after a sham surgery (sham) or an ovariectomy (OVX), ovariectomized Sprague-Dawley rats were placed on a diet containing 0 (OVX group) or 0.1% genistein for 4 weeks. The sham rats were fed a high-fat diet containing 0% genistein and served as the control group (sham group). The ovariectomized rats showed increases in body weight and insulin resistance index, but genistein reduced insulin resistance index and the activity of hepatic fatty acid synthetase. Genistein was also associated with increased activity of succinate dehydrogenase and carnitine palmitoyltransferase and the rate of β-oxidation in the fat tissue of rats. The ovariectomized rats given genistein had smaller-sized adipocytes. Using gene-set enrichment analysis (GSEA) of microarray data, we found that a number of gene sets of fatty acid metabolism, insulin resistance, and oxidative stress were differentially expressed by OVX and reversed by genistein. This systemic approach of GSEA enables the identification of such consensus between the gene expression changes and phenotypic changes caused by OVX and genistein supplementation. Genistein treatment could help reduce insulin resistance through the amelioration of OVX-induced metabolic dysfunction, and the GSEA approach may be useful in proposing putative targets related to insulin resistance.