This study was conducted to explore the mechanism by which caffeine increases GLUT4 expression in C2C12 myotubes. Myoblasts were differentiated in DMEM containing 2% horse serum for 13 days and the resultant myotubes exposed to 10 mM caffeine ± 25 µM KN93 or 10 mM dantrolene for 2 h. After the treatment, cells were kept in serum-free medium and harvested between 0 - 6 h later, depending on the assay. Chromatin immunoprecipitation (ChIP) assay revealed that caffeine treatment caused hyperacetylation of histone H3 at the MEF2 site on the Glut4 promoter (p < 0.05) and increased the amount of myocyte enhancer factor 2 (MEF2)-A that was bound to this site ~ 2.2-fold (p < 0.05) 4 h post treatment, compared to controls. These increases were accompanied by ~1.8-fold rise (p < 0.05; vs. control) in GLUT4 mRNA content at 6 h post caffeine treatment. Both immunoblot and immunocytochemical analyses showed reduced nuclear content of histone deacetylase-5 in caffeine-treated myotubes compared to controls at 0 - 2 h post treatment. Inclusion of 10 mM dantrolene in the medium to prevent the increase in cytosolic Ca²⁺, or 25 µM KN93 to inhibit Ca²⁺/ calmodulin-dependent protein kinase (CaMK II), attenuated all the above caffeine-induced changes. These data indicate that caffeine increases GLUT4 expression by acetylating the MEF2 site to increase MEF2A binding via a mechanism that involves CaMK II.