Rotating night shift work disrupts circadian rhythms and is associated with coronary heart disease. The relation between rotating night shift work and ischemic stroke is unclear. The Nurses' Health Study, an ongoing cohort study of registered female nurses, assessed in 1988 the total number of years the nurses had worked rotating night shifts. The majority (69%) of stroke outcomes from 1988 to 2004 were confirmed by physician chart review. The authors used Cox proportional hazards models to assess the relation between years of rotating night shift work and ischemic stroke, adjusting for multiple vascular risk factors. Of 80,108 subjects available for analysis, 60% reported at least 1 year of rotating night shift work. There were 1,660 ischemic strokes. Rotating night shift work was associated with a 4% increased risk of ischemic stroke for every 5 years (hazard ratio = 1.04, 95% confidence interval: 1.01, 1.07; P(trend) = 0.01). This increase in risk was similar when limited to the 1,152 confirmed ischemic strokes (hazard ratio = 1.03, 95% confidence interval: 0.99, 1.07; P(trend) = 0.10) and may be confined to women with a history of 15 or more years of rotating shift work. Women appear to have a modestly increased risk of stroke after extended periods of rotating night shift work.