Since the early 1970s, the agricultural frontier of southeastern Amazon has undergone extensive land use changes. These alterations, combined with regional climate changes, have the potential to influence the hydrologic cycle at small to large scales. We evaluated a 40-year time series (1976 to 2015) of rainfall and water yield and related them to land use changes in the Upper Xingu River Basin (UX). We acquired data from six rainfall stations and four river gauges and mapped land use changes. Mann-Kendall trend analysis and Pettitt’s change point detection were employed to describe annual and seasonal changes in the time series. Monthly water yield from the Xingu River was used to derive annual, seasonal, and monthly water yield, as well as the runoff coefficient. The largest changes in land use occurred during the last two decades and approximately 60,900 km2 in the Upper Xingu Basin were deforested between 1985 and 2015. Rainfall in the Xingu Basin decreased by about 245 mm over the period but there was no trend in water yield. The number of rainy days and intensity of events also decreased, but the length of the rainy season and seasonal and annual water yield did not change. Although watershed deforestation has increased water yield in other Amazon rivers, the reduction in rainfall in the Upper Xingu Basin was high enough to mask this effect.