Epidemiologic evidence supports an inverse association between physical activity and postmenopausal breast cancer. Whether associations exist for moderate activities, such as walking, and whether associations differ by estrogen receptor (ER) status, body mass index (BMI, kg/m(2)), adult weight gain, or use of postmenopausal hormones (PMH) is unclear. The relation between time spent sitting and breast cancer also is unclear. Among 73,615 postmenopausal women in the American Cancer Society Cancer Prevention Study II Nutrition Cohort, 4,760 women were diagnosed with breast cancer between 1992 and 2009. Extended Cox regression was used to estimate multivariable-adjusted relative risks (RR) of breast cancer in relation to total recreational physical activity, walking, and leisure-time sitting. Differences in associations by ER status, BMI, weight gain, and PMH use were also evaluated. The most active women (those reporting >42 MET-hours/week physical activity) experienced 25% lower risk of breast cancer than the least active [0-<7 MET-hours/week; 95% confidence interval (CI), 0.63-0.89; Ptrend = 0.01]. Forty-seven percent of women reported walking as their only recreational activity; among these women, a 14% lower risk was observed for ≥7 hours/week relative to ≤3 hours/week of walking (95% CI, 0.75-0.98). Associations did not differ by ER status, BMI, weight gain, or PMH use. Sitting time was not associated with risk. These results support an inverse association between physical activity and postmenopausal breast cancer that does not differ by ER status, BMI, weight gain, or PMH use. The finding of a lower risk associated with ≥7 hours/week of walking may be of public health interest.