Changes in environmental conditions could have an impact on genetically modified crops. The responses of photosynthetic physiological characteristics to CO2 in mature cotton leaves of two transgenic cotton varieties (transgenic Bt cotton Z30 and transgenic CpTI-Bt cotton SGK321) and their non-transgenic parental counterparts (Z16 and SY321) were investigated under different CO2 concentration conditions (0, 50, 75, 100, 200, 300, 400, 600, 800μmol?mol-1 and 1000μmol?mol-1) at squaring and boll-opening stages. The results showed that the parameters of apparent initial carboxylation efficiency, the maximum net photosynthetic rate and apparent dark respiration rate that calculated by model fitting in two transgenic cotton varieties were not significantly altered compared with those in parental counterparts. The obvious changes of responses of net photosynthetic rate in single leaf and water use efficiency to CO2 in Bt and CpTI-Bt transgenic cotton varieties were related to transgenic cotton genotypes and developmental stages at high CO2 concentration (above 700μmol?mol-1). At different developmental stages, no obvious differences in responses of stomatal conductance to CO2 between transgenic cotton varieties and their parental counterparts were observed, suggesting that higher CO2 concentration had no significant short-term effects on stomatal conductance of transgenic cottons.