We conducted a meta-analysis to summarize the evidence from prospective studies regarding the association between physical activity and breast cancer risk. A comprehensive search was conducted to identify eligible studies. The fixed or random effect model was used based on heterogeneity test. The dose-response relationship was assessed by restricted cubic spline model and multivariate random-effect meta-regression. Overall, 31 studies with 63,786 cases were included, and the combined relative risk (RR) with 95 % CI of breast cancer was 0.88 (0.85-0.91). In subgroup analysis by activity type, data from 27 studies including 37,568 cases for non-occupational activity (including recreational activity and household activity) and seven studies including 28,268 cases for occupational activity were used, and the RR (95 % CI) of breast cancer was 0.87 (0.83-0.91) and 0.90 (0.83-0.97), respectively. The inverse association was consistent among all subgroups analyses. Stronger association was found for subjects with BMI <25 kg/m(2) [0.72 (0.65-0.81)], premenopausal women [0.77 (0.72-0.84)], and estrogen and progesterone receptor-negative breast cancer [0.80 (0.73-0.87)]. Dose-response analysis suggested that the risk of breast cancer decreased by 2 % (P < 0.00) for every 25 metabolic equivalent (MET)-h/week increment in non-occupational physical activity, 3 % (P < 0.00) for every 10 MET-h/week (roughly equivalent to 4 h/week of walking in 2 miles/h or 1 h/week of running in 6 miles/h) increment in recreational activity, and 5 % (P < 0.00) for every 2 h/week increment in moderate plus vigorous recreational activity, respectively. Physical activity could significantly reduce the risk of breast cancer.