HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.