Women exhibit an enhanced capability for lipid metabolism during endurance exercise compared with men. The underlying regulatory mechanisms behind this sex-related difference are not well understood but may comprise signaling through a myocyte enhancer factor 2 (MEF2) regulatory pathway. The primary purpose of this study, therefore, was to investigate the protein signaling of MEF2 regulatory pathway components at rest and during 90 min of bicycling exercise at 60% Vo(2peak) in healthy, moderately trained men (n = 8) and women (n = 9) to elucidate the potential role of these proteins in substrate utilization during exercise. A secondary purpose was to screen for mRNA expression of MEF2 isoforms and myogenic regulatory factor (MRF) family members of transcription factors at rest and during exercise. Muscle biopsies were obtained before and immediately after exercise. Nuclear AMP-activated protein kinase-alpha (alphaAMPK) Thr(172) (P < 0.001), histone deacetylase 5 (HDAC5) Ser(498) (P < 0.001), and MEF2 Thr (P < 0.01) phosphorylation increased with exercise. No significant sex differences were observed at rest or during exercise. At rest, no significant sex differences were observed in mRNA expression of the measured transcription factors. mRNA for transcription factors MyoD, myogenin, MRF4, MEF2A, MEF2C, MEF2D, and peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC1alpha) were significantly upregulated by exercise. Of these, MEF2A mRNA increased 25% specifically in women (P < 0.05), whereas MEF2D mRNA tended to increase in men (P = 0.11). Although minor sex differences in mRNA expression were observed, the main finding of the present study was the implication of a joint signaling action of AMPK, HDAC5, and PGC1alpha on MEF2 in the immediate regulatory response to endurance exercise. This signaling response was independent of sex.