The association between fish, ω-3 and ω-6 polyunsaturated fatty acid (PUFA) intake and risk of colorectal cancer (CRC) remains inconclusive. Recent prospective studies suggest that the relationship may vary by gender, subsite and duration of follow-up. We followed 123,529 US adults (76,386 women and 47,143 men) without a history of cancer at baseline for 24 to 26 years. Fish and PUFA intake was assessed at baseline and updated every 4 years by using a validated food-frequency questionnaire. We found no overall association between fish, ω-3 and ω-6 PUFA intake and CRC risk with hazard ratio (HR) of 1.03 [95% confidence interval (CI): 0.89-1.20] comparing marine ω-3 intake of ≥ 0.30 g/d versus <0.15 g/d among women and 1.05 (95% CI: 0.85-1.30) comparing intake of ≥ 0.41 g/d versus <0.16 g/d among men. However, fish and marine ω-3 PUFA intake appeared to be positively associated with risk of distal colon cancer in both men and women and inversely with risk of rectal cancer in men. In an analysis based on a limited number of cases, marine ω-3 PUFA intake assessed 12-16 years before diagnosis tended to be inversely associated with CRC risk in men (HR: 0.76; 95% CI: 0.52-1.10). In conclusion, although no overall association between fish, ω-3 or ω-6 PUFA intake was observed with CRC risk, marine ω-3 PUFA may be differentially associated with risk of distal colon and rectal cancers and a long latency may be needed for its protection against CRC in men.