Changing ecological conditions along environmental gradients influence patterns of biodiversity and ecosystem functioning. However, how networks of interacting species respond to these changes remains unclear. We quantified aquatic food webs along longitudinal stream gradients spanning the Rocky Mountain-Great Plains ecotone using community composition, functional traits, and stable isotopes. We predicted that increasing ecosystem size, productivity, and species richness along the gradient would positively influence aquatic trophic diversity (e.g., expanded vertical and horizontal trophic niche breadths). We also predicted that trophic redundancy among fish species would decrease moving downstream as species partition food resources (e.g., reduced trophic niche overlap). Consumer stable isotope data (δ13 C and δ15 N) revealed nonlinear changes in trophic diversity along the gradient. Invertebrate trophic diversity had a dome-shaped relationship with the gradient, strongly linked to an expanding then contracting δ13 C range. Fish trophic diversity initially increased and then plateaued downstream, despite linearly expanding δ13 C and δ15 N ranges. Trophic redundancy within the fish community decreased downstream along the gradient. However, trophic redundancy also showed a nonlinear relationship with fish species richness; it initially declined, then began to increase when more than nine species were present, indicating a shift from niche partitioning to niche packing at intermediate species richness levels. This result suggests that while δ13 C and δ15 N ranges for fish communities increased across the gradient, niche packing within communities in the Great Plains caused overall trophic diversity to saturate. Our results demonstrate that food web structure along stream gradients reflects an interaction between factors that decrease trophic redundancy, such as increased living space and niche partitioning, versus factors that increase trophic redundancy, such as increased species richness and niche packing. Our study helps to explain how multiple mechanisms shape food web properties along longitudinal stream gradients, and where niche partitioning or niche packing may be dominant. Understanding the functional roles of organisms across similar environmental gradients in other ecosystems will be increasingly important because they determine how food webs, and thus ecosystem function, will respond to environmental change, biodiversity loss, or species invasions.