Animal and human data have suggested that shift work involving circadian disruption may be carcinogenic for humans, but epidemiological evidence for colorectal cancer remains limited. We investigated the association of rotating night shift work and colorectal cancer risk in two prospective female cohorts, the Nurses' Health Study (NHS) and NHS2, with 24 years of followup. In total, 190,810 women (NHS = 77,439; NHS2 = 113,371) were included in this analysis, and 1,965 incident colorectal cancer cases (NHS = 1,527; NHS2 = 438) were reported during followup (NHS: 19882012, NHS2: 19892013). We used Cox proportional hazards models adjusted for a wide range of potential confounders. We did not observe an association between rotating night work duration and colorectal cancer risk in these cohorts (NHS: 114 years: Hazard Ratio (HR) 1.04, 95% CI: 0.94, 1.16; 15+ years: HR 1.15, 95% CI: 0.95, 1.39; Ptrend = 0.14 and NHS2: 114 years: HR 0.81, 95% CI: 0.66, 0.99; 15+ years: HR 0.96, 95% CI: 0.56, 1.64 and Ptrend = 0.88). In subsite analysis in NHS, rectal cancer risk increased after longterm (15+ years) rotating night shift work (proximal colon cancer: HR 1.00, 95% CI: 0.75, 1.34, Ptrend = 0.90; distal colon cancer: HR 1.27, 95% CI: 0.87, 1.85, Ptrend = 0.32; rectal cancer: HR 1.60, 95% CI: 1.09, 2.34, Ptrend = 0.02). We found no overall evidence of an association between rotating night shift work and colorectal cancer risk in these two large cohorts of nurses. Risk for rectal cancer significantly increased with shift work duration, suggesting that longterm circadian disruption may play a role in rectal cancer development.