Purpose. This systematic review and meta-analysis was performed to determine the effectiveness of Omega-3 polyunsaturated fatty acid (n-3 PUFA) supplement on muscle soreness after eccentric exercise. Methods. PubMed, EMBASE, CENTRAL, and ISI Web of Science were searched to identify randomized controlled trials (RCTs) that assessed the efficacy of n-3 PUFA on muscle soreness after eccentric exercise. Mean difference (MD) and the associated 95% confidence interval (95% CI) were calculated by RevMan 5.3 to indicate delayed onset muscle soreness (DOMS) that measured two days after eccentric trainings. Subgroup analyses according to duration and daily dosage of n-3 PUFA supplements before eccentric exercises were performed to determine whether these factors will influence the overall effect size. The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) approach was used to evaluate the certainty of evidence. The protocol of this systematic review and meta-analysis was registered at PROSPERO (). Results. 12 RCTs containing 145 subjects and 156 controls were included in this study. Meta-analysis revealed a significantly decreased DOMS (MD -0.93; 95% CI -1.44, -0.42; P=0.0004) in n-3 PUFA supplement groups, while no significant differences in isometric muscle strength and range of motion (ROM) were detected. However, the pooled effect size for DOMS was lower than the minimal clinically important difference (MCID) of 1.4 on the 10-unit VAS, suggesting that the effect size of less muscle soreness with n-3 PUFA supplements did not appear to be clinically relevant. Conclusion. There is low-quality evidence that n-3 PUFA supplementation does not result in a clinically important reduction of muscle soreness after eccentric exercise. Isometric muscle soreness and range of motion were not improved by n-3 PUFA supplementation either (low-quality evidence). To further elucidate the overall role of n-3 PUFA on muscle damage in this area, large-scale RCTs are still needed.