Rivers are increasingly used as superhighways for the continental-scale transportation of freight goods, but the ecological impact of large vessel traffic on river ecosystems is difficult to study. Recently, the temporary maintenance closure of lock and dam systems on the Illinois Waterway (USA) brought commercial vessel traffic to a halt along the river's length, offering a rare opportunity to study the response of the ecosystem before, during, and after an extended pause of this persistent anthropogenic disturbance. We observed improvements in main- and side-channel water quality and a redistribution of fish habitat-use during a months-long, near-complete reduction of large vessel traffic. Over 3600 water quality and 1300 fish community samples indicate that large vessel traffic reduction coincided with a 33 % reduction in turbidity as well as increased use of sampling strata near vessel navigation corridors by sound-sensitive and rheophilic fishes. Gizzard shad (Dorosoma cepedianum), the most abundant species in the system, also expanded their use of these ‘impact’ areas. Though inland waterway transport is an economically- and climate-friendly alternative to trucking and rail for the shipment of freight, our data suggest that intense vessel traffic may have profound physical and biological impacts across a large river. Monitoring and mitigation of ecological impacts of the ongoing expansion of inland waterway transport around the world will be critical to balancing large rivers as both useful navigation corridors and functional ecosystems.