Background: The relationship between specific characteristics of physical activity (PA) (eg, intensity, type, frequency) with sex hormones is uncertain. The authors evaluated the association between characteristics of PA and circulating sex hormones. Methods: This was a cross-sectional analysis of the Women’s Lifestyle Validation Study (n = 493). Total PA, light-intensity PA (LPA), and moderate- to vigorous-intensity PA (MVPA) were assessed by accelerometry (a) and self-report (sr). Self-report was used to assess PA type (ie, aerobic, weight training) and exercise frequency. Dehydroepiandrosterone sulfate, testosterone, and sex hormone-binding globulin (SHBG) were assayed among all women; estradiol was assayed in postmenopausal women not currently on hormone therapy. Results: Estradiol was inversely associated and SHBG positively associated with MVPA and LPA (estradiol: β = −0.15 per SD increase, P ≤ .01 for a-MVPA and a-LPA; SHBG: a-MVPA β = 0.20 per SD increase, P ≤ .01, a-LPA β = 0.15, P < .01). By type, aerobic activity and weight training were each independently associated with estradiol and SHBG. Controlling for body mass index attenuated all associations for estradiol, and to a lesser extent SHBG. PA was not associated with testosterone levels. Conclusions: Multiple aspects of PA were independently associated with sex hormones; associations varied some by activity intensity and type, and were attenuated after accounting for body mass index.