The growing impact of human activities on the environment has increased their influence on the planet’s natural cycles, especially in relation to the hydrological cycle of watersheds. The fundamental processes for its water and energy balance have been affected, which influences water availability and surface streamflow. This study sought to evaluate the anthropogenic impacts on the hydrological cycle of the São Francisco River Basin (SFRB), Brazil, between 1985 and 2015. The study area comprised SFRB and 10 sub-basins for general and specific analyses, respectively. Analyzed data consisted of Land Use and Land Cover (LULC), precipitation, streamflow, and temperature. The methodology incorporated: (i) assessment of LULC dynamics; (ii) trend analysis with the Mann–Kendall method and Sen’s Slope; and (iii) decomposition of total streamflow variation via Budyko’s hypothesis and climate elasticity of streamflow. As a result, it was possible to detect an anthropic modification of SFRB, which is the main component of its streamflow variation, in addition to increased streamflow sensitivity to climate variations. In addition, the divergent behavior in the trends of hydrological variables suggests a change in the streamflow response to precipitation. Therefore, the results allowed us to identify and quantify the impacts of anthropic modifications on the hydrological cycle of the SFRB.