The potamodromous species worldwide has been threatened by dams intercepting large rivers. One strategy of mitigation is providing passage through fish ladders; however, ladders in Neotropical rivers have been considered as only ascending routes. We study the possible occurrence of and upstream and downstream movements through the fish ladder of Prochilodus lineatus at a large dam – Porto Primavera – in the heavily impounded Upper Paraná River, Brazil. A total of 1,419 specimens of P. lineatus were PIT-tagged in areas downstream (n = 807) and upstream (n = 612) of the dam, and continuously monitored for 4 years with the RFID system. We documented bidirectional movements of P. lineatus through the fish ladder. Of the 807 fish tagged downstream, 306 (37.9%) entered the ladder and 267 (87.2%) successfully reached the upstream exit. Conversely, of the 612 fish released upstream, 86 (14.0%) entered the ladder and 78 (90.7%) successfully reached the downstream exit. The entry proportion was significantly diferent between upstream and downstream (likelihood ratio chi-square: χ² = 104.7, p < 0.01) but not different between success likelihood ratio chi-square: χ² = 0.8, p = 0.37). Many individuals repeated these movements; one individual as many as six times. Besides, eight fish tagged in upstream probably moved to downstream by alternative routes (turbines, spillways or locks), because were not recorded descending the fish ladder but were recorded ascending. Thus, the 353 fish performed 452 passes through the fish ladder. This study showed the Porto Primavera fish ladder provides, bidirectional passage for P. lineatus. We suggest that fishways can acts as connectivity tools between upstream and downstream habitats.