This study investigated the physiological response of Populus euphratica (P. euphratica) to ecological water transport. Results showed significant increases in net photosynthetic (32.71%), stomatal conductance (27.58%), and transpiration (25.18%) rates of P. euphratica prior to the ecological water transport treatment. Internal CO2 concentrations (Ci) decreased significantly compared with the day preceding the treatment (23.69%; P < 0.05). During the treatment, the O, J, I, and P steps quickly increased, with the P step exhibiting the most significant change (P < 0.05). Moreover, Fv /Fm and Fv/Fo values were highest 7 d after the ecological water transport treatment. During the treatment, the initial fluorescence (F0), the maximal fluorescence intensity (Fm), PI, and RC/CSo quickly increased, with an increasing percentage of 9.67%, 46.15%, 59.17%, and 48.54%. In contrast, Vj, ABS/RC, TRo/RC, and ETo/RC rapidly decreased, with a decreasing percentage of 30.43%, 43.54%, 37.50%, and 39.04%, respectively. After the treatment, the average chlorophyll content of a, b, and a + b increased by 26.36%, 8.89%, and 21.93%, respectively, compared with the day preceding the treatment. This study also found that the relationship between soil water content and the net photosynthetic rate, stomatal conductance, the transpiration rate, the internal CO2 concentration, Fv/Fm, and Fv/Fo of P. euphratica were strongest during ecological water transport.