Context Hydrological and land use changes for human purposes, have resulted in the increased fragmentation of river landscapes and the loss of aquatic habitats, leading to profound changes in fish diversity and productivity. Objectives In the fluvial Lake Saint-Pierre (St. Lawrence River, Canada), we studied how agricultural practices and water-flow regulation have impacted the area and connectivity of spawning habitats of northern pike (Esox lucius). Methods Northern pike spawning and nursery habitats were modelled over a 49-year period (1965–2013) to estimate effective spawning area under four contrasting hydrological conditions. Results Simulations coupled with land cover analyses revealed that natural flow conditions historically favourable for fish reproduction (high and stable water flows) have been lost due to human activities. The highest potential for reproduction and habitat connectivity have been lost due to (1) intensive agriculture and ploughing of natural vegetation in the upper floodplain that overlaps suitable spawning areas for northern pike, and (2) flow regulation that has lowered and shortened spring floods and dried spawning grounds more frequently. Conclusions To restore the St. Lawrence River functions, we propose to reconvert the portion of the floodplains that is vital to fish, but is currently used by intensive agriculture, into natural wetlands or perennial crops and to restore a more natural flow regime by extending the duration of floods between spawning and nursery periods. The highest priority for habitat restoration should target the most effective and recurrent spawning habitats, ditch and stream networks, and connected managed wetlands.