We investigated the role played by CaMK II in the regulation of GLUT4 expression in response to intermittent exercise. Wistar rats completed 5 X 17 min bouts of swim exercise after receiving 5 mg/kg KN93 (an inhibitor of CaMK) or KN92 (an analog of KN93 which does not inhibit CaMK II); or an equivalent volume of vehicle. Triceps muscles that were harvested at 0, 6 or 18 h post exercise were assayed for: a) CaMK II phosphorylation by Western blot, b) acetylation of histone H3 at the MEF2 site on the Glut4 promoter by chromatin immunoprecipitation assay (ChIP), c) bound MEF2A at the Glut4 MEF2 cis element (ChIP) and d) GLUT4 expression by RT-PCR and Western blot. Compared to controls, there was a significant increase of ~ 100% in CaMK II phosphorylation after exercise and immunohistochemical stains indicated increased phosphorylation in nuclear and peri-nuclear regions of the muscle fiber. Acetylation of histone H3 in the region surrounding the MEF2 binding site on the Glut4 gene and the amount of MEF2A that bound to the site increased ~2-fold post exercise. GLUT4 mRNA and protein increased ~ 2.2-fold and ~2-fold, respectively, after exercise. The exercise-induced increases in histone H3 acetylation, MEF2A binding and GLUT4 expression were attenuated or abolished when KN93, was administered to rats prior to exercise. These data support the hypothesis that CaMK II activation by exercise increases GLUT4 expression via increased accessibility of MEF2A to its cis element on the gene.