The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role of PI-3-kinase and small GTP-binding proteins has been proposed. In previous studies we, among many others, excluded a role for the ras/MAP kinase pathway in insulin-mediated glucose transport. In this study we examined a possible role of the small GTP-binding protein rho in this process. Pretreatment of 3T3-L1 adipocytes with botulinum C3 exoenzyme (C3), which is known to ADP-ribosylate and inactivate rho, potently stimulated glucose uptake to a level similar to insulin. Interestingly, glycogen synthesis was not affected by C3 treatment. Insulin stimulates glucose uptake by triggering the translocation of GLUT4, the insulin-sensitive glucose transporter isotype, from an intracellular compartment to the plasma membrane. Similarly, C3-induced glucose uptake was paralleled by GLUT4 translocation. These data point to an important and novel role of the target of C3 (likely rho) in the regulation of GLUT4-mediated glucose transport. Our data suggest that insulin might stimulate glucose uptake through inactivation of rho.