The association between long-chain omega-3 polyunsaturated fatty acids (n-3 PUFA) and prostate cancer (PC) remains unclear. We compared incident PC rates as a function of the Omega-3 Index O3I, erythrocyte eicosapentaenoic and docosahexaenoic acids (EPA + DHA) in 5607 men (40-80 years of age) seen at the Cooper Clinic who were free of PC at baseline. The average follow-up was 5.1 ± 2.8 years until censoring or reporting a new PC diagnosis. Proportional hazards regression was used to model the linear association between baseline O3I and the age-adjusted time to diagnosis. A meta-analysis of n-3 PUFA biomarker-based studies and incident PC was updated with the present findings. A total of 116 cases of incident PC were identified. When O3I was examined as a continuous variable, the age-adjusted hazard ratio (HR) (95% CI) was 0.98 (0.89, 1.07; = 0.25) for each 1% increment in the O3I. The updated meta-analysis with 10 biomarker-based studies found no significant relationship between EPA or DHA levels and risk for PC. We find no evidence in this study nor in a meta-analysis of similar studies that consuming n-3 PUFA-rich fish or using fish oil supplements affects the risk of PC.