Abstract The mechanistic bases of thermal acclimation of net photosynthetic rate ( A n ) are still difficult to discern and empirical research remains limited, particularly for hybrid poplar. In the present study, we examined the contribution of a number of biochemical and biophysical traits on thermal acclimation of A n 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 10 weeks, we measured leaf RuBisCO and RuBisCO activase ( RCA ) amounts and the temperature response of A n , dark respiration ( R d ), stomatal conductance, ( g s ), maximum carboxylation rate of CO 2 ( V cmax ) and photosynthetic electron transport rate ( J ). Results showed that a 10°C increase in growth temperature resulted in a shift in thermal optimum ( T opt ) of A n of 6.2±1.6 °C and 8.0±1.2 °C for clone M×B and M×N respectively, and an increased A n and g s 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 RCA amount and the ratio of its short to long isoform was stimulated by warm condition for clone M×N and at low N for clone M×B. The activation energy of V cmax and J decreased under warm condition for clone M×B and remain unchanged for clone M×N. Our study demonstrated the involvement of both RCA , activation energy of V cmax and stomatal conductance in thermal acclimation of A n .