Glucose transporter 4 (Glut4) is an important regulator of cellular glucose uptake in adipose tissue and skeletal muscle. The estrogen receptors α and β (ERα and ERβ) have been shown to regulate Glut4. However, the regulatory mechanisms are unclear, and there are conflicting results about the effects of the two ER isoforms on Glut4 activity. In this study we investigated how the lack of either ER isoform affects Glut4 expression in differentiated mouse embryonic fibroblasts. Our results demonstrate that Glut4 transcription is markedly reduced in cells lacking ERβ, both basally and upon induction by liver X receptor. These changes in Glut4 expression could not be explained by the lack of ERβ as ligand-activated transcription factor. They were rather brought about by hypermethylation of one single CpG in the Glut4 promoter in the ERβ-deficient cells. This CpG is part of an Sp1-binding site, and Sp1 binding was reduced by its methylation. Treatment with Sp1 inhibitor diminished Glut4 expression in wild-type, but not in ERβ-deficient cells, suggesting that reduced recruitment of Sp1 to the Glut4 promoter is responsible for the differences in Glut4 expression. Reintroduction of ERβ into ERβ-deficient cells partly restored Glut4 transcription and stabilized low DNA methylation after treatment with the DNA demethylating agent 5-Aza-2'-deoxycytidine. Our findings demonstrate the involvement of DNA methylation in Glut4 regulation and imply a novel function for ERβ in mediating epigenetic events and thereby regulating gene expression.