Exercise/muscle contraction increases cell surface glucose transporter 4 (GLUT4), leading to glucose uptake to regulate blood glucose level. Elevating cytosolic Ca(2+) mediates this effect, but the detailed mechanism is not clear yet. We used calcium ionophore ionomycin to raise intracellular cytosolic Ca(2+) 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. siPKCalpha and siPKCtheta but not siPKCdelta and siPKCepsilon inhibited the ionomycin-increased cell surface GLUT4myc level. siPKCalpha, siPKCtheta inhibited the phosphorylation of AS160 and TBC1D1 induced by ionomycin. siPKCalpha and siPKCtheta prevented ionomycin-inhibited endocytosis of GLUT4myc. siPKCtheta, but not siPKCalpha inhibited ionomycin-stimulated exocytosis of GLUT4myc. siRab13 but not siRab8a, siRab10 and siRab14 inhibited the exocytosis of GLUT4myc promoted by ionomycin. In summary, ionomycin-promoted exocytosis of GLUT4 is partly reversed by siPKCtheta, whereas ionomycin-inhibited endocytosis of GLUT4 requires both siPKCalpha and siPKCtheta. PKCalpha and PKCtheta contribute to ionomycin-induced phosphorylation of AS160 and TBC1D1. Rab13 is required for ionomycin-regulated GLUT4 exocytosis.