During endurance exercise women have lower carbohydrate and higher lipid oxidation compared with men. Supplementation of humans and rodents with 17β-estradiol (E2) lowers the respiratory exchange ratio, the glucose rate of appearance and disappearance, and the metabolic clearance rate. The mechanism(s) for the observed estrogen effects in substrate utilization remains to be determined. We hypothesized that estrogen would increase the mRNA and protein content for genes involved in the regulation of β-oxidation. Ten moderately active men were supplemented with placebo or E2 for 8 days in a randomized double-blind crossover design. After supplementation muscle biopsies were obtained from the vastus lateralis and examined for differences in mRNA, microRNA, and protein content of genes involved in lipid oxidation. E2 increased the protein abundance of medium-chain acyl-CoA dehydrogenase (MCAD) 42% ( P ≤ 0.05). Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA was significantly higher after E2 supplementation by 29% ( P ≤ 0.05), and microRNA miR-29b (predicted to regulate PGC-1α) was significantly lower by 66% ( P ≤ 0.05). In conclusion, E2 might partially regulate lipid metabolism in skeletal muscle by altering the protein content of MCAD, which may be directly or indirectly regulated by an increase in PGC-1α and reduction in miR-29b.