The mechanistic bases of thermal acclimation of net photosynthetic rate (An) are still difficult to discern, and the data sets available are scarce, particularly for hybrid poplar. In the present study, we examined the contribution of a number of biochemical and biophysical traits on thermal acclimation of An for two hybrid poplar clones. We grew cuttings of Populus maximowiczii × Populus nigra (M×N) and Populus maximowiczii × Populus balsamifera (M×B) clones under two day/night temperature of 23°C/18°C and 33°C /27°C and under low and high soil nitrogen level. After ten weeks, we measured leaf RuBisCO (RAR) and RuBisCO activase (RARCA) amounts and the temperature response of An, dark respiration (Rd), stomatal conductance, (gs), apparent maximum carboxylation rate of CO2 (Vcmax) and apparent photosynthetic electron transport rate (J). Results showed that a 10°C increase in growth temperature resulted in a shift in thermal optimum (Topt) of An of 6.2±1.6°C and 8.0±1.2°C for clone M×B and M×N respectively, and an increased An and gs at the growth temperature for clone M×B but not M×N. RuBisCO amount was increased by N level but was insensitive to growth temperature while RARCA amount and the ratio of its short to long isoform was stimulated by the warm condition for clone M×N and at low N for clone M×B. The activation energy of apparent Vcmax and apparent J decreased under the warm condition for clone M×B and remained unchanged for clone M×N. Our study demonstrated the involvement of both RARCA, the activation energy of apparent Vcmax and stomatal conductance in thermal acclimation of An.