Objective: Multiple studies have shown clear evidence of vitamin D's anti-tumor effects on prostate cancer cells in laboratory experiments, but the evidence has not been consistent in humans. We sought to examine the association between vitamin D and prostate cancer risk in a cohort of older men. Methods: We conducted a prospective case-cohort study nested within the multicenter Osteoporotic Fractures in Men (MrOS) study. Baseline serum 25-OH vitamin D was measured in a randomly selected sub-cohort of 1,433 men ≥ 65 years old without a history of prostate cancer and from all participants with an incident diagnosis of prostate cancer (n = 297). Cox proportional hazards models were used to evaluate the associations between quartiles of total 25-OH vitamin D and incident prostate cancer, as well as Gleason score. Results: In comparison with the lowest quartile of 25-OH vitamin D, the hazard ratio for the highest quartile of 25-OH vitamin D was 1.22 (CI 0.50-1.72, p = 0.25), no trend across quartiles (p = 0.94) or association with Gleason score was observed. Adjustment for covariates did not alter the results. Conclusions: In this prospective cohort of older men, we found no association between serum 25-OH vitamin D levels and subsequent risk of prostate cancer. © 2010 The Author(s).