Widespread river regulation is known to modify river-aquifer interactions, influencing entire watersheds, but knowledge of the hyporheic flowpath along regulated rivers is limited. This study measured the hydraulic conductivity of the river bed and the aquifer, water levels and seepage fluxes in the heavily regulated Lule River in Northern Sweden, with the aim of characterising water exchange across the river-aquifer interface. While pristine rivers in the area are gaining, the Lule River was recharging the aquifer during 10% of the time. Daily river level fluctuations (typically ±0.25 m) directed ∼3% of the total orthogonal flux across the river bed towards the aquifer, while during ∼2% of the time the orthogonal fluxes were negligible (≤10–4 m d–1). A clogging layer on the river bed, most likely formed due to the modified river discharge, restricted river-aquifer exchange. The hyporheic zone had higher electrical conductivity than the river and the aquifer and electrical conductivity occasionally decreased following rising river water levels, with 3–5 hours' delay. Overall, hydropower regulation has severely altered the hydrological regime of the hyporheic zone in the Lule River.