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In arid regions, stream-flow regulation impacts the hydrological processes and riparian ecosystems in both direct and indirect ways. Thus, understanding the degree of eco-hydrological effects over different spatiotemporal scales is critical for effective stream-flow regulation. This study investigates the multiple hydro-ecological effects caused by a mandatory implementation of stream-flow regulation in a typical endorheic river basin, the Heihe River Basin (HRB), in Northwest China. The changes in the stream-flow regime, spatiotemporal patterns of vegetation and groundwater flow system were quantified using stream-flow data, leaf area index products of 1km resolution and a 3D numerical groundwater flow model. The results show that the stream leakage to groundwater is strongly correlated with the disturbance by stream-flow regulation, moderately with the evaporation and inflow to terminal lake, but only weakly with the total vegetation state and cropland change. Even though the average groundwater level for the entire lower HRB has an upward trend in response to increasing stream flow, it has no significant correlation with flow regulation. The groundwater system acts as a temporary bank to buffer the regulated stream flow for the entire lower HRB. The spatiotemporal patterns of the leaf area index distribution point to the stream-flow regulation as triggers for the increase in cropland and vegetation patches in the flat terrain along the river branches. This case study in the lower HRB has provided valuable database and insights for environmental flow management of other arid watersheds.