Variation in hydrological connectivity (HC) between rivers and lakes gives rise to periodic large water level fluctuation (WLF) in river-connected lakes (RCL). However, how WLF shapes the material cycling and concomitant trophodynamics of RCL remain unclear. Here we evaluated the resource availability, relative contributions of autochthonous and allochthonous carbon sources (by Bayesian mixing models) and trophic structure (using δ13C and δ15N) of Lake Poyang, the largest freshwater lake in China, during different hydrological periods. Terrestrial C3 plants in the aquatic/terrestrial transition zones (ATTZ) were the main supporter of aquatic consumers all year round. Compared with historical data, the contributions of autochthonous production to aquatic food webs were low. This is primarily ascribed to the influences of hydrological regimes in that the operation of Three Gorges Dam and HC changed the rhythm of WLF in Lake Poyang, restricting the production of aquatic vegetation and epiphytic algae. Most common fish species indicated higher trophic positions (TPs) during the dry than wet season, but only eleven of thirty-six common species showed a statistically significant increase (p < 0.05). Our study reveals a relatively consistent trophic diversity of consumer communities (fish and aquatic invertebrates) during dry and wet seasons, but each trophic guild showed specific trophic response to WLF, as they flexibly adjusted feeding strategies and the degree of trophic niche differentiation. Although WLF triggered fluctuations in the resource availability and species composition of lake food webs, and the TPs and trophic niches of aquatic consumers during different hydrological periods, the resource diversity and habitat complexity driven by HC and WLF reduced the strength of trophic interactions, and thus resulted in overall stability of food web trophodynamics and trophic structure of Lake Poyang. Our findings highlighted the importance of HC and natural WLFs in maintaining the ecosystem stability of large floodplain lakes, and that seasonal WLF patterns should be maintained in future floodplain lake management to conserve biodiversity and the resilience of ecological functions.