Photosynthetic and chlorophyll fluorescence characteristics of transgenic Bt Populus nigra seedlings were studied to discover whether there were unexpected effects of exogenous genes on photosynthesis of transgenic P. nigra. The results revealed that transgenic and non-transgenic P. nigra showed no statistically significant differences in photosynthetic parameters except for Intercellular CO2 concentration (Ci) and photochemical efficiency (Fv/Fm). The diurnal fluctuation of net photosynthetic rates of both transgenic and non-transgenic P. nigra is represented by a bimodal curve, but peak values occurred at different time between the two. Net photosynthetic rate of the transgenic P. nigra was significantly and positively correlated with Ci, Gs and Tr (P0.01), but in non-transgenic P. nigra only the curve of diurnal variation of Gs showed basically the same trend as those of photosynthetic rate. Photosynthetic “noon-break” of transgenic P. nigra was mainly caused by stomatal limitation, while “noon-break” of the control was limited by both stomatal and non-stomatal factors. Of the parameters of Pn-PFD response curves, only Rd showed a statistically significant difference, of which the Rd of control plants was 52.8% higher than that of the transgenic P. nigra. Thus, it is suggested that P. nigra had a higher physiological activity. Compared to the control, Fv/Fm of transgenic P. nigra declined enormously at 14:30 and did not recover to its normal level until 17:00, suggesting that photochemical efficiency was affected in the photosynthetic apparatus of P. nigra. We conclude that exogenous genes do not have a non-targeted effect on photosynthesis of transgenic P. nigra seedlings.