Sex steroid hormones, such as testosterone and estradiol, play important roles in developing both strength and mass of skeletal muscle. Recently, we demonstrated that skeletal muscle can synthesize sex steroid hormones. Whether there are sex differences in basal steroidogenesis or acute exercise-induced alterations of steroidogenesis in the skeletal muscle is unknown. We examine sex differences in the levels of testosterone, estradiol, and steroidogenesis-related enzymes, such as 17-hydroxysteroid dehydrogenase (HSD), 3-HSD, and aromatase cytochrome P450 (P450arom), in the skeletal muscle at rest and after exercise. We studied the gastrocnemius muscles of resting rats (10 weeks old) and exercised rats (10 weeks old, treadmill running, 30 m/min, 30 min). Basal muscular testosterone levels were higher in males than females, whereas estradiol did not differ between sexes. Additionally, 17-HSD, 3-HSD, and P450arom transcript and protein expression were greater in females. After acute exercise, testosterone levels and 17-HSD expression increased in muscle in both sexes. By comparison, muscular estradiol levels increased in males following exercise but were unchanged in females. Expression of P450arom, which regulates estrogen synthesis, increased after acute exercise in males, but decreased after exercise in females. Thus, a single bout of exercise can influence the steroidogenic system in skeletal muscle and these alterations differ between sexes. The acute exercise-induced alteration of steroidogenic enzymes may be enhance the locally steroidogenesis in the skeletal muscle in both sexes.