To investigate the trophic niche characteristics of fish in regulated lakes under water level fluctuations, Hongze Lake was selected as the study area. Carbon and nitrogen stable isotope techniques were used to analyze the trophic levels, trophic niche widths, niche overlap, and major food sources of six common benthic fish species—Cyprinus carpio, Pelteobagrus nitidus, Hemibarbus maculatus, Pelteobagrus fulvidraco, Saurogobio dabryi, and Paracanthobrama guichenoti—during the low-water period (July) and the high-water period (November). The results showed that the average trophic levels of all six benthic fish species ranged from 2.43 to 3.65, placing them at mid-trophic levels. However, their δ13C–δ1?N spatial distributions, trophic levels, and resource use patterns differed significantly between species and water-level periods. Distinct trophic niche characteristics were observed for the six fish species across the two periods. Specifically, the trophic niches of S. dabryi and P. guichenoti expanded significantly during the high-water period, indicating trophic niche expansion; the trophic niches of P. nitidus and P. fulvidraco contracted overall, showing trophic niche compression; whereas H. maculatus exhibited relatively stable trophic niche widths between the two periods but demonstrated trophic niche shifts. Niche overlap analysis revealed that trophic niche overlap among benthic fishes was highly asymmetric during the low-water period, suggesting stronger potential interspecific competition, while niche overlap became more symmetric during the high-water period, indicating a reduction in competitive pressure. These findings suggest that water level fluctuations, by altering habitat conditions and food resource patterns, significantly impact the trophic niche structure and interspecific relationships of benthic fishes in Hongze Lake. This study provides valuable insights into fish resource use and coexistence mechanisms under water level fluctuations, and offers scientific guidance for fish resource conservation in regulated lakes.