The purpose of this study was to investigate the potential role of creatine in GLUT4 gene expression in rat skeletal muscle. Female Wistar rats were fed with normal rat chow (controls) or chow containing 2% creatine monohydrate ad libitum for 3 weeks. GLUT4 protein levels of creatine fed rats were significantly increased in extensor digitorum longus (EDL), triceps, and epitrochlearis muscles compared to muscles from controls (p<0.05), and triceps GLUT4 mRNA levels were ~100% greater in triceps muscles from creatine-fed rats than in muscles from controls (P<0.05). In epitrochlearis muscles from creatine-fed animals, glycogen content was ~40% greater (P<0.05) and insulin-stimulated glucose transport rates were higher (P<0.05) than in epitrochlearis muscles from controls. Despite no changes in [ATP], [creatine], [phosphocreatine], or [AMP], creatine feeding increased AMP-activated protein kinase (AMPK) phosphorylation by 50% in rat EDL muscle (p<0.05). Creatinine content of EDL muscle was almost 2-fold higher for creatine-fed animals than for controls (P<0.05). Creatine feeding increased protein levels of myocyte enhancer factor 2 (MEF2) isoforms, MEF2A (~70%, p<0.05), MEF2C (~60%, p<0.05) and MEF2D (~90%, p<0.05), which are transcription factors that regulate GLUT4 expression, in creatine-fed rat EDL muscle nuclear extracts. Electrophoretic mobility shift assay showed that DNA binding activity of MEF2 was increased by ~40% (p<0.05) in creatine-fed rat EDL compared to control. Our data suggest that creatine feeding enhances the nuclear content and DNA binding activity of MEF2 isoforms, which is concomitant with an increase in GLUT4 gene expression.