Exercise/muscle contraction increases cell surface glucose transporter 4 (GLUT4), leading to glucose uptake to regulate blood glucose level. Elevating cytosolic Ca2+ mediates this effect, but the detailed mechanism is not clear yet. We used calcium ionophore ionomycin to raise intracellular cytosolic Ca2+ level to explore the underlying mechanism. We showed that in L6 myoblast muscle cells stably expressing GLUT4myc, ionomycin increased cell surface GLUT4myc levels and the phosphorylation of AS160, TBC1D1. siPKC alpha and siPKC theta but not siPKC8 and siPKCe inhibited the ionomycin-increased cell surface GLUT4myc level. siPKC alpha, siPKC theta inhibited the phosphorylation of AS160 and TBC1D1 induced by ionomycin. siPKC alpha and siPKCO prevented ionomycin-inhibited endocytosis of GLUT4myc. siPKC theta, but not siPKC alpha inhibited ionomycin-stimulated exocytosis of GLUT4myc. siRabl3 but not siRab8 alpha, siRabl theta and siRabl4 inhibited the exocytosis of GLUT4myc promoted by ionomycin. In summary, ionomycinpromoted exocytosis of GLUT4 is partly reversed by siPKC theta, whereas ionomycin-inhibited endocytosis of GLUT4 requires both siPKC alpha and siPKC theta. PKC alpha and PKC theta contribute to ionomycin-induced phosphorylation of AS160 and TBC1D1. Rab13 is required for ionomycin-regulated GLUT4 exocytosis. (C) 2017 Elsevier Inc. All rights reserved.