Background: Several studies have linked vasectomy with the risk of prostate cancer; however, this association has been attributed to selection bias. Since vasectomy is a common and effective form of contraception, these implications are significant. Therefore, we sought to test this association in a large observational cohort. Objective: To evaluate the potential association between prior vasectomy and the risk of developing prostate cancer. Materials and Methods: We evaluated the relationship between vasectomy and prostate cancer in the NIH-AARP Diet and Health Study. Of the 111,914 men, prostate cancer was identified in 13,885 men and vasectomies were performed in 48,657. We used multivariate analysis to examine the relationship between prostate cancer and vasectomy. We also performed propensity score-adjusted and propensity score-matched analysis. Results: Men utilizing vasectomy were more likely to be ever married, fathers, educated, white, and screened for prostate cancer. During 4,251,863 person-years of follow-up, there was a small association between vasectomy and incident prostate cancer with a hazard ratio of 1.05 (95% CI, 1.01-1.11). However, no significant association was found when looking separately at prostate cancer by grade or stage. Conclusions were similar when using propensity adjustment and matching. Importantly, a significant interaction between vasectomy and PSA screening was identified. Discussion: Estimates of the association between vasectomy and prostate cancer are sensitive to analytic method underscoring the tenuous nature of the connection. Given the differences between men who do and do not utilize vasectomy, selection bias appears likely to explain any identified association between vasectomy and prostate cancer. Conclusions: With over 20 years of follow-up, no convincing relationship between vasectomy and prostate cancer of any grade was identified.