Habitat fragmentation is an important cause of biodiversity loss in freshwater systems, as worldwide rivers have been fragmented by dams and other hydraulic structures. To restore freshwater fish populations, some barriers have been removed, but the long-term ecological effects of this removal have been rarely quantified. In the present study, we quantified the effects of barrier removal on river colonization by anadromous sea lamprey (Petromyzon marinus) by analyzing the spatial distribution and nest density in a small coastal river (France) from 1994 to 2011. Our results demonstrated the benefit of dam removal within few years after restoration. Indeed, the spatial distribution of nests shifted significantly upstream and was more uniform throughout the river after removal. Our results also suggest that the spatial patterns of habitat colonization were affected by the density of nests, river flow and connectivity. Finally, although the number of nests was significantly higher after removal, it was not possible to clearly identify the contribution of intrinsic versus external factors involved in this pattern. Further investigations are therefore needed to quantify the potential subsequent effects on juvenile recruitment and the overall population dynamics. Copyright (c) 2014 John Wiley & Sons, Ltd.