Overexpression of GLUT4 exclusively in skeletal muscle enhances insulin action and improves glucose homeostasis. Transgenic studies have discovered two regions on the GLUT4 promoter conserved across several species that are required for normal GLUT4 expression in skeletal muscle. These regions contain binding motifs for the myocyte enhancer factor 2 (MEF2) family and GLUT4 enhancer factor (GEF). A single bout of exercise increases both GLUT4 transcription and mRNA abundance; however, the molecular mechanisms mediating this response remain largely unexplored. Thus, the aim of this study was to determine whether a single, acute bout of exercise increased the DNA-binding activities of MEF2 and GEF in human skeletal muscle. Seven subjects performed 60 min of cycling at approximately 70% of VO2peak. After exercise, the DNA-binding activities of both the MEF2A/D heterodimer and GEF were increased (P<0.05). There was no change in nuclear MEF2D or GEF abundance after exercise, but nuclear MEF2A abundance was increased (P<0.05). These data demonstrate that exercise increases MEF2 and GEF DNA binding and imply that these transcription factors could be potential targets for modulating GLUT4 expression in human skeletal muscle.