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.