Increasing rates of biodiversity loss are adding momentum to efforts seeking to restore or rewild degraded landscapes. Here, we investigated the effects of natural forest regeneration on water flux partitioning, water ages and hydrological connectivity, using the tracer-aided ecohydrological model EcH2O-iso. The model was calibrated using ¯3.5 years of diverse ecohydrological and isotope data available for a catchment in the Scottish Highlands, an area where impetus for native pinewood regeneration is growing. We then simulated two land cover change scenarios that incorporated forests at early (dense thicket) and late (old open forest) stages of regeneration, respectively. Changes to forest structure (proportional vegetation cover, vegetation heights and leaf area index of pine trees) were modelled for each stage. The scenarios were then compared to a present-day baseline simulation. Establishment of thicket forest had substantial ecohydrological consequences for the catchment. Specifically, increased losses to transpiration and, in particular, interception evaporation drove reductions in below-canopy fluxes (soil evaporation, groundwater (GW) recharge and streamflow) and generally slower rates of water turnover. The greatest reductions in streamflow and connectivity were simulated for summer baseflows and small to moderate events during summer and the autumn/winter rewetting period. This resulted from the effect of local changes to flux partitioning in regenerating areas on the hillslopes extending to the wider catchment by reducing downslope GW subsidies that help sustain summer baseflows and saturation in the valley bottom. Meanwhile, higher flows were relatively less affected, especially in winter. Despite the generally drier state of the catchment, simulated water ages suggested that the increased transpiration demands of the thicket forest could be satisfied by moisture carried over from previous seasons. The more open nature of the old forest generally resulted in water fluxes, water ages and connectivity returning towards baseline conditions. Our work implies that the ecohydrological consequences of natural forest regeneration depend on the structural characteristics of the forest at different stages of development. Consequently, future land cover change investigations need to move beyond consideration of simple forest vs. non-forest scenarios to inform sustainable landscape restoration efforts.