Glucose transporter 4 (GLUT4), a major contributor to glucose transport in skeletal muscle, is closely related to diabetic treatment. Exercise regulates apparently GLUT4 gene expression which produces many beneficial metabolic adaptations for diabetic patients. Study has shown that both AMPK (AMP-activated protein kinase) and CaMK (calcium-calmodulin dependent protein kinase) signaling pathways are involved in regulation of exercise-induced GLUT4 gene expression in skeletal muscles, but the relationship between these two signaling pathways in regulating the GLUT4 gene is unclear. The purpose of the following study was to investigate the relationship of these two pathways in Caffeine- and AICAR-stimulated skeletal muscle cells GLUT4 gene expression. Muscle contractile activity results in increases in both cytosolic Ca²⁺ and AMPK activity. To mimic this response, primary cultured rat skeletal muscle cells were treated with Caffeine to raise cytosolic Ca²⁺ and with AICAR to activate AMPK. The muscle cells were divided into different groups (Control, AICAR, Caffeine, AICAR/Caffeine, Caffeine+Compound C, AICAR/Caffeine+Compound C, AICAR+KN93, AICAR/Caffeine+KN93), which were used for experiments of stimulation by AICAR and Caffeine, inhibition by AMPK inhibitor, Compound C and CaMK inhibitor, KN93 respectively. The results showed that both AICAR and Caffeine induced about 2- and 3-fold increases respectively (P < 0.05, AICAR vs. Control; P < 0.05, Caffeine vs. Control) in GLUT4 mRNA in muscle cells, but the effect of GLUT4 mRNA induced by the combined stimulation of raising cytosolic Ca²⁺ and activating AMPK was not additive. Moreover, the Compound C, an AMPK inhibitor, decreased the Caffeine-induced increases in GLUT4 mRNA (P < 0.05, Caffeine+Compound C vs. Caffeine), and also attenuated an increase in GLUT4 mRNA induced by the combined stimulation of AICAR and Caffeine (P < 0.05, AICAR/Caffeine+Compound C vs. AICAR/Caffeine). Similarly, the Caffeine induced an increase in α1-AMPK phosphorylation (P < 0.05, Caffeine vs. Control) and furthermore the Compound C reduced apparently such an increase (P < 0.05, Caffeine+Compound C vs. Caffeine); however, KN93, a CaMK inhibitor, was completely able to inhibit the Caffeine-induced increase in GLUT4 mRNA, but failed to inhibit the AICAR-induced 2-fold increases in GLUT4 mRNA (P < 0.05, AICAR+KN93 vs. Control) and also failed to block an increase in GLUT4 mRNA induced by the combined stimulation of AICAR and Caffeine (P < 0.05, AICAR+Caffeine+KN93 vs. Control). These results demonstrate that the CaMK and AMPK signaling pathways are not complete independent, but are cooperative in the regulation of Caffeine-induced GLUT4 gene expression in cultured skeletal muscle cells. Collectively, based on our results and the other results this paper suggests that the GLUT4 gene expression in contracting skeletal muscle cells may be regulated through a CaMK-AMPK signaling pathway.