AbstractNearly half of recent decades' global forest loss occurred in the Amazon and Cerrado (tropical savanna) biomes of Brazil, known as the arc of deforestation. Despite prior analysis in individual river basins, a generalizable empirical understanding of the effect of deforestation on streamflow across this region is lacking. We frame land use change in Brazil as a natural experiment and draw on in situ and remote sensing evidence in 324 river basins covering more than 3 × 106 km2 to estimate streamflow changes caused by deforestation and agricultural development between 1950 and 2013. Deforestation increased dry season low flow by between 4 and 10 percentage points (relative to the forested condition), corresponding to a regional‐ and time‐averaged rate of increase in specific streamflow of 1.29 mm/year2, equivalent to a 4.08 km3/year2 increase, assuming a stationary climate. In conjunction with rainfall and temperature variations, the net (observed) average increase in streamflow over the same period was 0.76 mm/year2, or 2.41 km3/year2. Thus, net increases in regional streamflow in the past half century are 58% of those that would have been experienced with deforestation given a stationary climate. This study uses a causal empirical analysis approach novel to the water sciences to verify the regional applicability of prior basin‐scale studies, provides a proof of concept for the use of observational causal identification methods in the water sciences, and demonstrates that deforestation masks the streamflow‐reducing effects of climate change in this region.