In the context of global warming attributable to the increasing levels of CO₂, severe drought may be more frequent in areas that already experience chronic water shortages (semiarid areas). This necessitates research on the interactions between increased levels of CO₂ and drought and their effect on plant photosynthesis. It is commonly reported that ¹³C fractionation occurs as CO₂ gas diffuses from the atmosphere to the substomatal cavity. Few researchers have investigated ¹³C fractionation at the site of carboxylation to cytoplasm before sugars are exported outward from the leaf. This process typically progresses in response to variations in environmental conditions (i.e., CO₂ concentrations and water stress), including in their interaction. Therefore, saplings of two typical plant species (Platycladus orientalis and Quercus variabilis) from semiarid areas of northern China were selected and cultivated in growth chambers with orthogonal treatments (four CO₂ concentration ([CO₂])  ×  five soil volumetric water content (SWC)). The δ¹³C of water-soluble compounds extracted from leaves of saplings was determined for an assessment of instantaneous water use efficiency (WUE_cp) after cultivation. Instantaneous water use efficiency derived from gas-exchange measurements (WUE_ge) was integrated to estimate differences in δ¹³C signal variation before leaf-level translocation of primary assimilates. The WUE_ge values in P. orientalis and Q.  variabilis both decreased with increased soil moisture at 35–80 % of field capacity (FC) and increased with elevated [CO₂] by increasing photosynthetic capacity and reducing transpiration. Instantaneous water use efficiency (iWUE) according to environmental changes differed between the two species. The WUE_ge in P. orientalis was significantly greater than that in Q. variabilis, while an opposite tendency was observed when comparing WUE_cp between the two species. Total ¹³C fractionation at the site of carboxylation to cytoplasm before sugar export (total ¹³C fractionation) was species-specific, as demonstrated in the interaction of [CO₂] and SWC. Rising [CO₂] coupled with moistened soil generated increasing disparities in δ¹³C between water-soluble compounds (δ¹³C_WSC) and estimates based on gas-exchange observations (δ¹³C_obs) in P. orientalis, ranging between 0.0328 and 0.0472 ‰. Differences between δ¹³C_WSC and δ¹³C_obs in Q. variabilis increased as [CO₂] and SWC increased (0.0384–0.0466 ‰). The ¹³C fractionation from mesophyll conductance (g_m) and post-carboxylation both contributed to the total ¹³C fractionation that was determined by δ¹³C of water-soluble compounds and gas-exchange measurements. Total ¹³C fractionation was linearly dependent on stomatal conductance, indicating that post-carboxylation fractionation could be attributed to environmental variation. The magnitude and environmental dependence of apparent post-carboxylation fractionation is worth our attention when addressing photosynthetic fractionation.