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