Water deficit is one of the key factors that limits the carbon (C) assimilation and productivity of plants. The effect of variable water deficit on recently root-derived bicarbonate assimilation in Camptotheca acuminate seedlings was investigated. Three-month-old seedlings were subjected to three water regimes, well-watered (WW), moderate stress (MS), and severe stress (SS) induced by polyethyleneglycol, in conjunction with relatively high (H) and low (L) natural C-13-abundance of NaHCO3-labeled treatments in hydroponics for 14 days. The delta C-13 of the newly expanded leaves in H were generally more enriched in heavy isotopes than were those in L, indicative of the involvement of bicarbonate in aboveground tissues. The C isotope fractionation of newly expanded leaves relative to air (a dagger C-13(air-leaves)) ranged from 17.78 to 21.78aEuro degrees among the treatments. The a dagger C-13(air-leaves) under the MS and SS treatments in H were both more negative than was that in L. A linear regression between Ci/Ca and a dagger C-13(air-leaves) in both L and H were different from the theoretical regression. On the basis of the two end-member mixing model, the proportion of fixed CO2 supplied from bicarbonate contributing to the total photosynthetically inorganic C assimilation were 10.34, 20.05 and 16.60% under the WW, MS, and SS treatments, respectively. These results indicated that the increase in water deficit decreased the atmospheric CO2 gain but triggered a compensatory use of bicarbonate in C. acuminate seedlings.