The Rio Grande is the fourth longest river in North America extending over 3,000 km from the Rocky Mountains to the Gulf of Mexico. The Pleistocene evolution of this river from individual subbasins into a coalesced fluvial system has been long debated. Herein, we constrain the middle Pleistocene evolution of the northernmost and largest Rio Grande basin, the San Luis basin, and the timing of incision of the Rio Grande Gorge, based on new geologic mapping, ³He surface exposure dating, and U-series dating on pedogenic carbonates. ³He dating of shoreline deposits of closed basin Lake Alamosa and fluvially scoured bedrock along the Rio Grande gorge rim indicate the San Luis basin was connected with southerly Rio Grande basins and progressively incised since ∼400 ka. Waning tectonic activity, coupled with the hydrologic response to intensified middle Pleistocene glaciation, drove southward spillover of Lake Alamosa ∼400 ka, expanding the river's drainage basin by nearly 24,000 km², and adding recharge areas in the high-altitude, glaciated San Juan, Tusas, and Sangre de Cristo Mountains. Geologic barriers to streamflow on the Taos Plateau were breached and basin tributary drainages were integrated and inset into their present canyons ∼250-200 ka, with approximately 120 m of incision during ∼400-200 ka. Previous work demonstrates that southerly streamflow through basins south of the SLB terminated into a large, rift-related bolson complex until the middle Pleistocene, and most axial Rio Grande river incision has occurred since 630 ka. We propose that middle Pleistocene integration of the SLB was the major geomorphic event that eroded Plio-Pleistocene tectonic and volcanic barriers and re-established flow of the Rio Grande from the Rocky Mountains to the Gulf of Mexico.