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Reference evapotranspiration (ETo) is an important hydrometeorological term widely used in understanding and projecting the hydrological effects of future climate and land use change. We conducted a case study in the Qinhuai River Basin that is dominated by a humid subtropical climate and mixed land uses in southern China. Long-term (1961-2012) meteorological data were used to estimate ETo by the FAO-56 Penman-Monteith model. The individual contribution from each meteorological variable to the trend of ETo was quantified. We found basin-wide annual ETo decreased significantly (p < 0.05) by 3.82 mm/yr during 1961-1987, due to decreased wind speed, solar radiation, vapor pressure deficit (VPD), and increased relative humidity (RH). However, due to the increased VPD and decreased RH, the ETo increased significantly (p < 0.05) in spring, autumn, and annually at a rate of 2.55, 0.56, and 3.16 mm/yr during 1988-2012, respectively. The aerodynamic term was a dominant factor controlling ETo variation in both two periods. We concluded the key climatic controls on ETo have shifted as a result of global climate change during 1961-2012. The atmospheric demand, instead of air temperature alone, was a major control on ETo. Models for accurately predicting ETo and hydrological change under a changing climate must include VPD in the study region. The shifts of climatic control on the hydrological cycles should be considered in future water resource management in humid regions.