Elevating cytosolic Ca²⁺ stimulates glucose uptake in skeletal muscle, but how Ca²⁺ affects intracellular traffic of GLUT4 is unknown. In tissue, changes in Ca²⁺ leading to contraction preclude analysis of the impact of individual, Ca²⁺-derived signals. In L6 muscle cells stably expressing GLUT4myc, the Ca²⁺ ionophore ionomycin raised cytosolic Ca²⁺ and caused a gain in cell surface GLUT4myc. Extra- and intracellular Ca²⁺ chelators (EGTA, BAPTA-AM) reversed this response. Ionomycin activated calcium calmodulin kinase II (CaMKII), AMPK, and PKCs, but not Akt. Silencing CaMKIIδ or AMPKα1/α2 partly reduced the ionomycin-induced gain in surface GLUT4myc, as did peptidic or small molecule inhibitors of CaMKII (CN21) and AMPK (Compound C). Compared with the conventional isoenzyme PKC inhibitor Gö6976, the conventional plus novel PKC inhibitor Gö6983 lowered the ionomycin-induced gain in cell surface GLUT4myc. Ionomycin stimulated GLUT4myc exocytosis and inhibited its endocytosis in live cells. siRNA-mediated knockdown of CaMKIIδ or AMPKα1/α2 partly reversed ionomycin-induced GLUT4myc exocytosis but did not prevent its reduced endocytosis. Compared with Gö6976, Gö6983 markedly reversed the slowing of GLUT4myc endocytosis triggered by ionomycin. In summary, rapid Ca²⁺ influx into muscle cells accelerates GLUT4myc exocytosis while slowing GLUT4myc endocytosis. CaMKIIδ and AMPK stimulate GLUT4myc exocytosis, whereas novel PKCs reduce endocytosis. These results identify how Ca²⁺-activated signals selectively regulate GLUT4 exocytosis and endocytosis in muscle cells.