We examined the association between omega-3 fatty acids (O3FAs) and prostate-specific antigen (PSA) in a cross-sectional analysis of 6219 men examined at the Cooper Clinic from 2009 to 2013. We assayed O3FAs from red blood cell membranes and measured PSA levels in study participants. Multiple logistic regression was used to examine the association between O3FAs and PSA. The mean age of study participants was 55.5 years (SD = 9.8) with a mean PSA level of 1.31 ng/mL (SD = 1.5). Unadjusted analyses indicated that there was a slight, direct association with PSA and each of the O3FAs tested. However, after adjusting for age and body mass index (BMI), the associations were reversed but nonsignificant [odds ratio (OR) for PSA > 4 ng/mL: total omega-3 OR = 0.98 per each 1% of total fatty acids, 95% confidence interval (CI) = 0.93-1.03; docosahexaenoic acid OR = 1.01, 95% CI = 0.92-1.11; omega-3 index OR = 0.99, 95% CI = 0.93-1.05). Similar results were obtained after age and BMI adjustment when the omega-3 index was divided into undesirable (0.01-3.99%), intermediate (4.0-7.99%), and desirable ranges ( ≥ 8.0%). Given that the study had >80% power to detect an odds ratio <0.9 or >1.1, we conclude that associations between O3FAs and PSA levels are either nonexistent or quite weak in the population that this healthy sample represents.