In adipocytes, insulin stimulates glucose transport principally by promoting translocation of glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Requirements for Ca2+/calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca2+ in this process is unknown. Recently, myosin II, whose function in non-muscle cells is primarily regulated by phosphorylation of its regulatory light chain (RLC) by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), was implicated in insulin-stimulated GLUT4 translocation. We have investigated, using 3T3-L1 and 3T3-F442A adipocytes, the possibility that MLCK may be involved in the insulin-stimulated translocation of GLUT4. Insulin significantly increases phosphorylation of the myosin II RLC in a Ca2+-dependent manner. ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and 2,3-butanedione monoxime, block insulin-stimulated GLUT4 translocation and subsequent glucose transport. In addition, suppression of MLCK expression via stably expressing antisense-MLCK decreases insulin-stimulated glucose transport. Our studies strongly suggest that MLCK may be a regulatory target of Ca2+/calmodulin and may play an important role in insulin-stimulated GLUT4 translocation in adipocytes. [J Physiol Sci. 2006;56 Suppl:S17]