Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle specific transporter (GLUT4). Therefore, our objective was to determine the effects of the contraction mediated signals, calcium and AMP activated protein kinase (AMPK), on glucose uptake and GLUT4 expression under acute and chronic conditions. To accomplish this, we utilized pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum Ca2+ releasing agent, had a biphasic effect on GLUT4 expression and glucose uptake. Low concentration (1.25 to 2 mM) or short term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide 1--D-ribonucleoside (AICAR), had an additive effect on GLUT4 expression. However, high concentration (2.5 to 5 mM) or long term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK induced GLUT4 expression in skeletal muscle.