To investigate the association between intake of fish and n-3 polyunsaturated fatty acids (n-3 PUFA) and the risk of breast cancer and to evaluate the potential dose-response relation. Meta-analysis and systematic review of prospective cohort studies. PubMed and Embase up to December 2012 and references of retrieved relevant articles. Prospective cohort studies with relative risk and 95% confidence intervals for breast cancer according to fish intake, n-3 PUFA intake, or tissue biomarkers. Twenty six publications, including 20,905 cases of breast cancer and 883,585 participants from 21 independent prospective cohort studies were eligible. Eleven articles (13,323 breast cancer events and 687,770 participants) investigated fish intake, 17 articles investigated marine n-3 PUFA (16,178 breast cancer events and 527,392 participants), and 12 articles investigated alpha linolenic acid (14,284 breast cancer events and 405,592 participants). Marine n-3 PUFA was associated with 14% reduction of risk of breast cancer (relative risk for highest v lowest category 0.86 (95% confidence interval 0.78 to 0.94), I(2)=54), and the relative risk remained similar whether marine n-3 PUFA was measured as dietary intake (0.85, 0.76 to 0.96, I(2)=67%) or as tissue biomarkers (0.86, 0.71 to 1.03, I(2)=8%). Subgroup analyses also indicated that the inverse association between marine n-3 PUFA and risk was more evident in studies that did not adjust for body mass index (BMI) (0.74, 0.64 to 0.86, I(2)=0) than in studies that did adjust for BMI (0.90, 0.80 to 1.01, I(2)=63.2%). Dose-response analysis indicated that risk of breast cancer was reduced by 5% per 0.1g/day (0.95, 0.90 to 1.00, I(2)=52%) or 0.1% energy/day (0.95, 0.90 to 1.00, I(2)=79%) increment of dietary marine n-3 PUFA intake. No significant association was observed for fish intake or exposure to alpha linolenic acid. Higher consumption of dietary marine n-3 PUFA is associated with a lower risk of breast cancer. The associations of fish and alpha linolenic acid intake with risk warrant further investigation of prospective cohort studies. These findings could have public health implications with regard to prevention of breast cancer through dietary and lifestyle interventions.