It has long been debated whether changes in Ca2+ are involved in insulin-stimulated glucose uptake in skeletal muscle. We have now investigated the effect of insulin on the global free myoplasmic Ca2+ concentration and the near-membrane free Ca2+ concentration ([Ca2+]mem) in intact, single skeletal muscle fibers from mice by using fluorescent Ca2+ indicators. Insulin has no effect on the global free myoplasmic Ca2+ concentration. However, insulin increases [Ca2+]mem by approximately 70% and the half-maximal increase in [Ca2+]mem occurs at an insulin concentration of 110 microunits per ml. The increase in [Ca2+]mem by insulin persists when sarcoplasmic reticulum Ca2+ release is inhibited but is lost by perfusing the fiber with a low Ca2+ medium or by addition of L-type Ca2+ channel inhibitors. Thus, insulin appears to stimulate Ca2+ entry into muscle cells via L-type Ca2+ channels. Wortmannin, which inhibits insulin-mediated activation of glucose transport in isolated skeletal muscle, also inhibits the insulin-mediated increase in [Ca2+]mem. These data demonstrate a new facet of insulin signaling and indicate that insulin-mediated increases in [Ca2+]mem in skeletal muscle may underlie important actions of the hormone.