The actin-cytoskeleton-regulating GTPase Rac1 is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Rac1 and its downstream signaling in glucose transport in insulin sensitive and insulin resistant mature skeletal muscle has not previously been investigated. We hypothesized that Rac1 and its downstream target, p21-activated kinase (PAK), are regulators of insulin-stimulated glucose uptake in mouse and human skeletal muscle, and are dysregulated in insulin resistant states.Muscle specific inducible Rac1 knockout (KO) mice and pharmacological inhibition of Rac1 were used to determine whether Rac1 regulates insulin-stimulated glucose transport in mature skeletal muscle. Furthermore, Rac1 and PAK1 expression and signalling were investigated in muscle of insulin resistant mice and humans.Inhibition and KO of Rac1 decreased insulin-stimulated glucose transport in mouse soleus and EDL muscles ex vivo. Rac1 KO mice showed decreased insulin and glucose tolerance and trended towards higher plasma insulin concentrations following intraperitoneal glucose injection. Rac1 protein expression and PAK(Thr423) phosphorylation were decreased in muscles of high fat fed mice. In humans, insulin-stimulated PAK-activation was decreased in both acute insulin resistant (intralipid infusion) and in chronic insulin resistant states (obesity and diabetes). These findings show that Rac1 is a regulator of insulin-stimulated glucose uptake and a novel candidate involved in skeletal muscle insulin resistance.