A protective effect of physical activity (PA) on breast cancer (BC) risk has been suggested. Few studies have examined the influence of PA on mammographic breast density (MBD), a strong risk factor for BC. In a prospective study in Florence, Italy, we identified 2,000 healthy women with a mammogram taken 5 years after enrollment. Individual mammograms were retrieved (83%) and MBD assessed according to Wolfe's classification. Detailed information on PA at work and during leisure time, reproductive history, lifestyle and anthropometric measurements at enrollment were available for 1,666 women. Information on hormone replacement therapy (HRT) was also obtained at mammogram. Women with high-MBD (P2 + DY Wolfe's patterns) were compared with women with low-MBD (N1 + P1) by multivariate logistic models. Overall, high-MBD was inversely associated with increasing levels of leisure time PA (p for trend = 0.04) and among peri-/postmenopausal women, also with increasing levels of recreational activities (p for trend = 0.02). An interaction between PA and HRT emerged, with a stronger inverse association of highest level of recreational activity with MBD among HRT nonusers (p for interaction = 0.02). A modifying effect by body mass index (BMI) was evident among 1,025 peri-/postmenopausal women who did not use HRT at the time of mammogram, with a stronger inverse association between recreational PA and MBD in the highest BMI tertile (OR = 0.34; 95% CI 0.20-0.57; p for interaction = 0.03). This large study carried out in Mediterranean women suggests that leisure time PA may play a role in modulating MBD, particularly in overweight/obese peri-/postmenopausal women.