Receptor-interacting protein 140 (RIP140), a nuclear receptor corepressor, is important for lipid and glucose metabolism. In adipocytes, RIP140 can be phosphorylated by protein kinase C epsilon (PKCvarepsilon), followed by arginine methylation, and exported to the cytoplasm. This study demonstrates for the first time a cytoplasmic function for RIP140: to counteract insulin-stimulated glucose transporter 4 (GLUT4) membrane partitioning and glucose uptake in adipocytes. Cytoplasmic RIP140 interacts with the Akt substrate AS160, thereby impeding AS160 phosphorylation by Akt; this in turn reduces GLUT4 trafficking. This signal transduction pathway can be recapitulated in the epididymal adipocytes of diet-induced obese mice: nuclear PKCvarepsilon is activated, cytoplasmic RIP140 increases, and GLUT4 trafficking and glucose uptake are reduced. The data reveal a new, cytoplasmic function for RIP140 as a negative regulator of GLUT4 trafficking and glucose uptake, and shed insight into the regulation of basal and insulin-stimulated glucose disposal by a nuclear-initiated counteracting mechanism.