Bladder cancer is the sixth most common cancer in the United States. Night shift work has previously been linked with cancer risk. Whether there is an association between rotating night shift work and bladder cancer in women has not been studied previously. Eligible participants in the Nurses' Health Study (NHS, n = 82,147, 1988-2016) and Nurses' Health Study II (NHSII, n = 113,630, 1989-2015) were prospectively followed and a total of 620 and 122 incident bladder cancer cases were documented during the follow-up of NHS and NHSII, respectively. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) for bladder cancer incidence. We observed a significantly increased risk of bladder cancer among women with >5 years of night shift work history compared with women who never worked rotating night shifts in NHS (HR = 1.24; 95%CI = 1.01-1.54, p for trend = 0.06), but not in the pooled NHS and NHS II (HR = 1.18; 95%CI = 0.97-1.43, p for trend = 0.08). Secondary analyses stratified by smoking status showed no significant interaction ( p = 0.89) between the duration of rotating night shift work and smoking status. In conclusion, our results did not provide strong evidence for an association between rotating night shift work and bladder cancer risk.