Human immunodeficiency virus (HIV) infection is associated with insulin resistance. HIV type 1 Nef downregulates cell surface protein expression, alters signal transduction, and interacts with the cytoskeleton and proteins involved in actin polymerization. These functions are required for glucose uptake by insulin-stimulated adipocytes. We sought to determine whether Nef alters adipocyte glucose homeostasis. Using radiolabeled glucose, we found that adipocytes exposed to recombinant Nef took in 42% less glucose after insulin stimulation than did control cells. This reduction resulted from a Nef-dependent inhibition of glucose transporter 4 (GLUT4) trafficking, as assessed by means of immunofluorescence microscopy. Immunoblot analysis revealed a decrease in phosphorylation of signal transducing proteins after Nef treatment, and fluorescence microscopy showed a dramatic alteration in cortical actin organization. We conclude that Nef interferes with insulin-stimulated processes in adipocytes. We have identified HIV Nef, which is detectable and antigenic in serum samples from HIV-infected people, as a novel contributor to the development of insulin resistance.