Research suggests that long-chain omega-3 polyunsaturated fatty acids (LC-PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have antineoplastic properties, yet evidence for association between LC-PUFAs and colorectal cancer (CRC) remains inconsistent. Using the VITamins And Lifestyle (VITAL) cohort, we evaluated how EPA/DHA intake, and its primary sources, fish oil supplement use and dark fish consumption, relate to CRC risk. A total of 68,109 Washington residents aged 50-76 completed a questionnaire between 2000-2002 and were followed for CRC through 2008 (n = 488). Persons using fish oil supplements on 4+ days/wk for 3+ yr experienced 49% lower CRC risk than nonusers (hazard ratio = 0.51, 95% CI = 0.26-1.00; P trend = 0.06). The association between fish oil use and decreased CRC risk was primarily observed for men (P interaction = 0.02; P trend men = 0.02; P trend women = 0.88) and for colon cancer (P difference = 0.05; P trend colon = 0.03; P trend rectum = 0.87). Although dark fish and total EPA + DHA intake were not associated with CRC risk overall, these associations varied by genetic risk (P interaction = 0.009 and 0.02, respectively), with inverse associations observed among low-moderate genetic risk groups and positive associations observed among high risk groups. Results suggest that associations between LC-PUFA intake and CRC may vary by gender, subsite, and genetic risk, providing additional insight into the potential role of LC-PUFAs in cancer prevention.