Iron homeostasis in marine organisms operates under chronically low iron bioavailability, which may shape the strategies of iron uptake and storage in fish. Stable iron isotope ratios (<em>δ</em><sup>56</sup>Fe) have emerged as tracers of iron storage and uptake in terrestrial mammals, yet the physiological drivers of isotope fractionation in marine organisms remain poorly understood. Here, we investigated <em>δ</em>⁵⁶Fe variation and iron speciation across eight tissues of wild chub mackerel (<em>Scomber japonicus</em>), along with pool size estimation of key Fe species, including ferritin-bound (ferric) and heme-bound (mainly ferrous) Fe, using Fe K-edge X-ray Absorption Near Edge Structure (XANES) spectroscopy. The liver <em>δ</em>⁵⁶Fe values were consistently higher than those of other tissues, with an apparent isotopic shift between ferritin- and heme-bound Fe (<em>Δ</em>⁵⁶Fe) in the liver averaging 2.04 ± 0.22 ‰ (2 S.D.). In contrast to the liver, no significant enrichment of heavy Fe isotope was observed in the ovary and red muscle despite their high ferritin-Fe contribution, suggesting a high interconversion rate between ferritin- and heme-bound Fe pools in these tissues. The overall range of <em>δ</em>⁵⁶Fe variation among tissues was smaller than that reported in mammals. Our results indicated that muscular <em>δ</em>⁵⁶Fe in marine teleost is primarily governed by source signatures and intestinal uptake efficiency, while tissue heterogeneity due to heavy Fe storage by ferritin exerts only a minor influence. These findings highlight the potential of <em>δ</em>⁵⁶Fe as a proxy for intestinal iron acquisition in fish and provide new geochemical perspectives on iron cycling through marine food webs.