Night work involving disruption of circadian rhythm was suggested as a possible cause of breast cancer. We examined the role of night work in a large population-based case-control study carried out in France between 2005 and 2008. Lifetime occupational history including work schedules of each night work period was elicited in 1,232 cases of breast cancer and 1,317 population controls. Thirteen percent of the cases and 11% of the controls had ever worked on night shifts (OR = 1.27 [95% confidence interval = 0.99–1.64]). Odds ratios were 1.35 [1.01–1.80] in women who worked on overnight shifts, 1.40 [1.01–1.92] in women who had worked at night for 4.5 or more years, and 1.43 [1.01–2.03] in those who worked less than three nights per week on average. The odds ratio was 1.95 [1.13–3.35] in women employed in night work for >4 years before their first full-term pregnancy, a period where mammary gland cells are incompletely differentiated and possibly more susceptible to circadian disruption effects. Our results support the hypothesis that night work plays a role in breast cancer, particularly in women who started working at night before first full-term pregnancy.