Temporal variations in reference evapotranspiration (ETo) have profound implications for hydrological processes as well as for agricultural crop performance. The main aim of this study was to analyze the annual, seasonal trends in ETo in the Heihe River basin. The likely causative meteorological variables for such temporal changes in ETo were also identified. Results showed that, on a seasonal and annual scale, ETo for the upper reach showed increasing trends from 1960 to 2010; both increasing and decreasing trends were observed for the middle and lower reaches. In spring, wind speed (WS) and relative humidity (RH) were the most likely causative variables for changes of ETo for the whole basin; in summer and autumn, maximum temperature (Tmax) and RH contributed more to the trends in ETo for the upper reach, and WS contributed more for the middle and lower reaches; in winter, Tmax, WS and RH contributed more in different locations and in different seasons. From the spatial perspective, WS, RH and Tmax contributed more to the changes of ETo in the upper reach; WS was the main likely influence factor in the middle reach, and WS and RH were the probable main factors in the lower reach.