Overweight and obesity are associated with increased postmenopausal breast cancer risk; however, it is unclear whether losing excess weight will lower risk. Therefore, we examined the relationship between weight loss and postmenopausal breast cancer among 13,055 overweight and obese, cancer-free women who enrolled in the Cancer Prevention Study-II (CPS-II) Nutrition Cohort in 1992. During the 15 year follow-up, 816 postmenopausal breast cancer cases were diagnosed. Self-reported weight was collected before diagnosis at baseline and 10 years prior to baseline. The median weight loss was 11 lbs, but only 58% of the women maintained this weight loss through the first 5 year follow-up interval (1992-1997). Using both restricted cubic splines and multivariate Cox proportional hazards modeling, we observed no association between weight loss and postmenopausal breast cancer. The hazard ratio for 30+ pounds of weight loss compared to stable weight was 0.95 (95%: CI 0.47-1.95). An inverse association was, however, suggested among women who maintained ten or more pounds of weight loss through the next interval. There was no evidence of effect modification by postmenopausal hormone use, initial BMI, or other factors examined. In summary, weight loss was not associated with postmenopausal breast cancer in this study. Future studies should focus on sustained weight loss and whether the timing of weight loss is important.