Background: The protective effect of physical activity on breast cancer risk might be mediated by sex hormone levels. In this study, we examined the association between usual physical activity and plasma levels of estrogens, androgens, dehydroepiandrosterone sulphate (DHEAS), and sex hormone binding globulin (SHBG) in postmenopausal women. Methods: We conducted a cross-sectional study among 806 postmenopausal women participating in the Prospect-EPIC study. Usual physical activity was assessed using a short questionnaire and summarized into a simple, validated four-level index. This index combines occupational physical activity with time spent on cycling and sporting. Levels of estrone, estradiol, androstenedione, DHEAS, testosterone, and SHBG were measured in plasma. General linear models were used to examine the association between usual physical activity and sex hormone levels, adjusted for confounders.