Water scarcity is a key challenge to global development. In Brazil, the Sao Francisco River Basin (SFB) has experienced water scarcity problems because of decreasing streamflow and increasing demands from multiple sectors (e.g., food and energy production and urban supply). However, the drivers of decreased streamflow, particularly the potential role of groundwater withdrawals, have not been yet investigated. Here, we assess long-term trends in baseflow, quickflow, and streamflow of the SFB during 1980–2015 and constrain the most likely drivers of observed decreases through trend analysis of precipitation (P), evapotranspiration (ET), and terrestrial water storage change (TWS). We found that over 82% of the observed decrease in streamflow can be attributed to a significant decreasing baseflow trend (< -20 m3 s-1 y-1) along the SFR with spatial agreement between decreased baseflow, increased ET, and irrigated agricultural land. We also found a decrease in TWS across the SFB with trends exceeding -20 mm y-1. Overall, our findings indicate that decreasing groundwater contributions (i.e., baseflow) are the primary cause of the observed reduction in total SFR flow. A lack of significant climate trends indicates that climate variability likely has not caused the observed baseflow reduction, mainly in the Middle and Sub-middle SFB, and therefore groundwater and surface withdrawals may be the most likely driver of water scarcity over the SFB. We call for increased attention on groundwater’s key role for the SFB and integrated regional management of surface and groundwater.