The conservation of potamodromous species worldwide has been threatened by the loss of longitudinal connectivity caused by dams intercepting large rivers. One environmental management strategy for reestablishing connectivity is providing passage through fish ladders. However, ladders in Neotropical rivers have been described as ascending one-way routes. We analyzed the movements of Prochilodus lineatus through a fish ladder at a large dam – Porto Primavera – in the heavily impounded Upper Paraná River, Brazil, to determine whether the ladder connected habitats downstream and upstream of the dam, in both directions. A total of 1,419 specimens of P. lineatus were PIT-tagged in areas downstream and upstream of the dam, and continuously monitored for 4 years. We documented bidirectional movements of P. lineatus through the fish ladder. Many individuals repeated these movements annually; one individual as many as six times. Thus, our study suggests the Porto Primavera fish ladder contributes to habitat connectivity, bidirectional passage, and conservation of P. lineatus. Our results deviate from the perception that fishways are ineffective in Neotropical rivers. Our data suggest that fishways can restore the bidirectional connectivity denied to some Neotropical species, and until the services of dams are no longer needed, environmental management through fish ladders may continue to be part of broader conservation strategy designed to preserve native fauna.