Endurance exercise (EE) leads to beneficial alterations in skeletal muscle lipid metabolism in overweight and obese individuals, however the mechanisms of these improvements are poorly understood. The primary goal of the current investigation was to test the hypothesis that long-term EE training (six months) leads to alterations in the mRNA abundance of key lipid metabolism enzymes in skeletal muscle of overweight and obese middle-aged women and men. A secondary aim of this study was to investigate the hypothesis that exercise-mediated adaptations in mRNA levels differ between women and men. The mRNA abundance of representative lipogenic and lipolytic genes from major lipid metabolism pathways, as well as representative lipogenic and lipolytic transcription factors, were determined by real-time PCR from skeletal muscle biopsies collected before and approximately 24 hours after the final bout of six months of EE. Six months of EE led to increases in muscle lipoprotein lipase (LPL), peroxisome proliferators-activated receptor co-activator-1 (PGC-1), carnitine palmitoyltransferase-1 (CPT-1), diacylglycerol acyltransferase-1 (DGAT-1), and acid ceramidase (AC) mRNA in women, but not men. In contrast, in men, EE led to reductions in the mRNA content of the lipogenic factors sterol regulatory element binding protein-1c (SREBP-1c) and serine palmitoyl transferase (SPT). These data suggest that EE-mediated alterations in the abundance of the lipid metabolism genes studied here are fundamentally different between overweight and obese middle-aged women and men. Future studies should determine whether these adaptations in mRNA levels translate into changes in protein function.