In general, fish residing in rivers differ from fish residing in lakes in their mercury (Hg) isotope ratios. Specifically, fish residing in lakes typically show enriched values for the isotope ratios of δ²⁰²Hg (mass-dependent fractionation of isotope ²⁰²Hg) and Δ¹⁹⁹Hg (mass-independent fractionation of isotope ¹⁹⁹Hg) compared with fish residing in rivers, because photochemical effects acting on Hg isotope ratios are stronger in lakes than in rivers. Whole-fish determinations of Hg isotope ratios in age-0 and adult (ages 4–11) walleye (Sander vitreus) caught in the Fox River, the main tributary to lower Green Bay of Lake Michigan, were dissimilar. Age-0 fish exhibited a river signature for δ²⁰²Hg and Δ¹⁹⁹Hg, with means equal to 0.00 and 0.26‰, respectively. Significantly elevated levels of δ²⁰²Hg and Δ¹⁹⁹Hg were observed in adult fish, indicating that adult fish primarily resided in the bay. Our results implied that the Fox River serves as a nursery area for juvenile walleye in the Fox River–lower Green Bay ecosystem. Moreover, corrections for photochemical fractionation of δ²⁰²Hg revealed that age-0 and adult walleye shared the same source of Hg in this ecosystem. In addition, Hg isotope ratios did not significantly differ between the sexes of adult walleye, suggesting that these ratios did not fractionate during the Hg elimination process.