Changes in sex hormone levels with aging or illness may lead to metabolic disorders. Moreover, the ratio changes in men versus women may have distinct pathological responses. Since little is known about sex hormone action on muscle metabolism, we examined the role of testosterone or 17b-estradiol (E2) in metabolism and investigated whether either hormone may mediate a sex-specific effect. Myotubes from postmenopausal women and age-matched male donors were treated with 10 nM testosterone or E2 for 4 days, and assays were performed to measure metabolic readouts, signal transduction, and mRNA expression. Testosterone and E2 treatment enhanced insulin-stimulated glucose incorporation into glycogen and AKT phosphorylation in myotubes from female donors, highlighting a sexspecific role of sex hormone in glucose metabolism. Testosterone treatment increased palmitate oxidation in myotubes from both female and male donors, while E2 enhanced palmitate oxidation in myotubes from male donors only. Testosterone-mediated increase in palmitate oxidation was attenuated at the presence of androgen receptor antagonist, which may indicate a role of nuclear steroid receptor in muscle lipid oxidation. Testosterone treatment increased mRNA expression of the insulin receptor substrate 2 in myotubes from male and female donors, whereas it increased mRNA expression of glycogen synthase 1 only in myotubes from male donors. E2 treatment increased pyruvate dehydrogenase kinase 4 mRNA expression in myotubes from female donors. Thus, our data suggest that testosterone or E2 modulates muscle glucose and lipid metabolism and may play a role in metabolism in a sex-dependent manner.