Insulin resistance is a common metabolic abnormality in women with PCOS and leads to an elevated risk of type 2 diabetes. Studies have shown that thiazolidinediones (TZDs) improve metabolic disturbances in PCOS patients. We hypothesized that the effect of TZDs in PCOS is, in part, mediated by changes in the transcriptional profile of muscle favoring insulin sensitivity. Using Affymetrix microarrays, we examined the effect of pioglitazone (30 mg/day for 16 weeks) on gene expression in skeletal muscle of 10 obese women with PCOS metabolically characterized by a euglycemic-hyperinsulinemic clamp. Moreover, we explored gene expression changes between these PCOS patients before treatment and 13 healthy women. Treatment with pioglitazone improved insulin-stimulated glucose metabolism and plasma adiponectin, and reduced fasting serum insulin (all P<0.05). Global pathway analysis using Gene Map Annotator and Pathway Profiler (GenMAPP 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1) revealed a significant upregulation of genes representing mitochondrial oxidative phosphorylation (OXPHOS), ribosomal proteins, mRNA processing reactome, translation factors, and proteasome degradation in PCOS after pioglitazone therapy. Quantitative real-time PCR suggested that upregulation of OXPHOS genes was mediated by an increase in PGC-1alpha expression (P<0.05). Pretreatment expression of genes representing OXPHOS and ribosomal proteins was down-regulated in PCOS patients compared to healthy women. These data indicate that pioglitazone therapy restores insulin sensitivity, in part, by a coordinated upregulation of genes involved in mitochondrial OXPHOS and ribosomal protein biosynthesis in muscle in PCOS. These transcriptional effects of pioglitazone may contribute to prevent the onset of type 2 diabetes in these women.