Night shift work has been classified by the International Agency for Research on Cancer as a probable carcinogen in humans. Several studies have assessed night shift work in relation to breast cancer risk, with inconsistent results. In the prospective Sister Study cohort, current and past occupational history was collected for 48,451 participants. We used Cox proportional hazards models to estimate adjusted HRs and 95% confidence intervals (CI) for the association between baseline work schedule characteristics and incident breast cancer. During follow-up (mean = 9.1 years), 3,191 incident cases were diagnosed. We observed little to no increase in risk associated with work schedule characteristics (ever working rotating shifts: HR = 1.04, 95% CI, 0.91-1.20; ever working rotating night shifts: HR = 1.08, 95% CI, 0.92-1.27; ever working at night: HR = 1.01, 95% CI, 0.94-1.10; and ever working irregular hours: HR = 0.98, 95% CI, 0.91-1.06). Although short-term night work (>0 to 5 years vs. never: HR = 1.12; 95% CI, 1.00-1.26) and rotating shift work at night (>0 to 5 years vs. never: HR = 1.30; 95% CI, 1.05-1.61) were associated with increased breast cancer risk, working nights for more than 5 years was not associated with risk. Overall, we observed little evidence that rotating shift work or work at night was associated with a higher risk of breast cancer, except possibly among those who participated in such work for short durations of time. This study indicates that if night shift work is associated with breast cancer, the increase in risk is small.