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1. Dams disrupt the river continuum, altering hydrology, biodiversity and energy flow. Although research indicates that tributary inputs have the potential to dilute these effects, knowledge at the food-web level is still scarce.

2. Here, we examined the riverine food-web structure of the Colorado River below Glen Canyon Dam, focusing on organic matter sources, trophic diversity and food chain length. We asked how these components respond to pulsed flows from tributaries following monsoon thunderstorms that seasonally increase streamflow in the American Southwest.

3. Tributaries increased the relative importance of terrestrial organic matter, particularly during the wet season below junctures of key tributaries. This contrasted with the algal-based food-web present immediately below Glen Canyon Dam.

4. Tributary inputs during the monsoon also increased trophic diversity and food chain length: food chain length peaked below the confluence with the largest tributary (by discharge) in Grand Canyon, increasing by >1 trophic level over a 4-5 km reach possibly due to aquatic prey being flushed into the mainstem during heavy rain events.

5. Our results illustrate that large tributaries can create seasonal discontinuities, influencing riverine food-web structure in terms of allochthony, food-web diversity and food chain length.

6. Synthesis and applications. Pulsed flows from unregulated tributaries following seasonal monsoon rains increase the importance of terrestrially derived organic matter in large, regulated river food webs, increasing food chain length and trophic diversity downstream of tributary inputs. Protecting unregulated tributaries within hydropower cascades may be important if we are to mitigate food-web structure alteration due to flow regulation by large dams. This is critical in the light of global hydropower development, especially in megadiverse, developing countries where dam placement (including completed and planned structures) is in tributaries.