Aberrant DNA methylation is associated with diabetes, but the precise regulatory events that control the levels and activity of DNA methyltransferases (DNMTs) is not well understood. Here we show that miR-539-5p targets Dnmt3b and regulates its cellular levels. miR-539-5p and Dnmt3b show inverse patterns of expression in skeletal muscle of diabetic mice. By binding to the 3' UTR of Dnmt3b, miR-539-5p downregulates its levels in C2C12 cells and in human primary skeletal muscle cells. miR-539-5p-Dnmt3b interaction regulates Srebf1 transcription by altering methylation at CpG islands within Srebf1 in C2C12 cells. Dnmt3b inhibition alone was sufficient to upregulate Srebf1 transcription. In vivo antagonism of miR-539-5p in normal mice induced hyperglycemia and hyperinsulinemia and impaired oral glucose tolerance. These mice had elevated Dnmt3b and decreased Srebf1 levels in skeletal muscle. db/db mice injected with miR-539-5p mimics showed improved circulatory glucose and cholesterol levels. Oral glucose tolerance improved together with normalization of Dnmt3b and Srebf1 levels in skeletal muscle. Our results support a critical role of miR-539-5p and Dnmt3b in aberrant skeletal muscle metabolism during diabetes by regulating Srebf1 transcription; modulating the miR-539-5p-Dnmt3b axis might have therapeutic potential for addressing altered skeletal muscle physiology during insulin resistance and type 2 diabetes.