Abstract Increased physical activity has been shown to be protective for breast cancer although few studies have examined this association in Black women. In addition, limited evidence to date indicates that sedentary behavior may be an independent risk factor for breast cancer. We examined sedentary behavior and physical activity in relation to subsequent incident breast cancer in a nested case–control study within 546 cases (374 among Black women) and 2,184 matched controls enrolled in the Southern Community Cohort Study. Sedentary and physically active behaviors were assessed via self-report at study baseline (2002–2009) using a validated physical activity questionnaire. Conditional logistic regression was used to estimate mutually adjusted ORs and corresponding 95% confidence intervals (CI) for quartiles of sedentary and physical activity measures in relation to breast cancer risk. Being in the highest versus lowest quartile of total sedentary behavior (≥12 vs. <5.5 h/d) was associated with increased odds of breast cancer among White women [OR, 1.94 (95% CI, 1.01–3.70); Ptrend = 0.1] but not Black women [OR, 1.23 (95% CI, 0.82–1.83); Ptrend = 0.6] after adjustment for physical activity. After adjustment for sedentary activity, greater physical activity was associated with reduced odds for breast cancer among White women (Ptrend = 0.03) only. In conclusion, independent of one another, sedentary behavior and physical activity are risk factors for breast cancer among White women. Differences in these associations between Black and White women require further investigation. Reducing sedentary behavior and increasing physical activity are potentially independent targets for breast cancer prevention interventions. Cancer Prev Res; 6(6); 566–76. ©2013 AACR.