OBJECTIVE: Low inflammation associated with fish intake may reflect not only the amount of fish consumed but also its nutrient composition and related lifestyle factors. This study investigated the relationship between fish intake, estimated n-3 polyunsaturated fatty acid (n-3 PUFA) intake, lifestyle behaviors, and the systemic immune-inflammation index (SII), a composite marker of atherosclerotic cardiovascular disease (ASCVD). METHODS: We conducted a cross-sectional study of 8675 participants (mean age: 46.7 ± 13.0 years; 59% men) undergoing health check-ups between April 2019 and March 2020. Fish intake was assessed using a self-reported questionnaire on weekly consumption frequency. Estimated weekly fish and n-3 PUFA intake were calculated by combining intake frequency with age- and sex-specific average intake data derived from the Japanese National Health and Nutrition Survey. RESULTS: The average fish intake was 143 ± 88 g/week. Higher fish intake was significantly associated with lower SII levels (p < 0.0001). In multivariable regression analysis, higher fish intake and aerobic exercise were independently associated with lower SII, whereas shorter sleep duration was associated with higher SII. The proportion of participants engaging in aerobic exercise increased across fish intake categories. Mediation analysis suggested that estimated n-3 PUFA intake substantially contributed to the association between fish intake and lower SII. CONCLUSION: The anti-inflammatory effects associated with fish intake may be partly explained by n-3 PUFA intake, along with related lifestyle factors. These findings highlight the importance of considering fish consumption alongside broader dietary and behavioral patterns that influence systemic inflammation.