Rotating night shift work disrupts circadian rhythms and has been associated with obesity, metabolic syndrome, and glucose dysregulation. However, its association with type 2 diabetes remains unclear. Therefore, we aimed to evaluate this association in two cohorts of US women. We followed 69,269 women aged 42-67 in Nurses' Health Study I (NHS I, 1988-2008), and 107,915 women aged 25-42 in NHS II (1989-2007) without diabetes, cardiovascular disease, and cancer at baseline. Participants were asked how long they had worked rotating night shifts (defined as at least three nights/month in addition to days and evenings in that month) at baseline. This information was updated every 2-4 years in NHS II. Self-reported type 2 diabetes was confirmed by a validated supplementary questionnaire. We documented 6,165 (NHS I) and 3,961 (NHS II) incident type 2 diabetes cases during the 18-20 years of follow-up. In the Cox proportional models adjusted for diabetes risk factors, duration of shift work was monotonically associated with an increased risk of type 2 diabetes in both cohorts. Compared with women who reported no shift work, the pooled hazard ratios (95% confidence intervals) for participants with 1-2, 3-9, 10-19, and ≥20 years of shift work were 1.05 (1.00-1.11), 1.20 (1.14-1.26), 1.40 (1.30-1.51), and 1.58 (1.43-1.74, p-value for trend <0.001), respectively. Further adjustment for updated body mass index attenuated the association, and the pooled hazard ratios were 1.03 (0.98-1.08), 1.06 (1.01-1.11), 1.10 (1.02-1.18), and 1.24 (1.13-1.37, p-value for trend <0.001). Our results suggest that an extended period of rotating night shift work is associated with a modestly increased risk of type 2 diabetes in women, which appears to be partly mediated through body weight. Proper screening and intervention strategies in rotating night shift workers are needed for prevention of diabetes.