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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 (<i>Platycladus orientalis</i> and <i>Quercus variabilis</i>) 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 <i>δ</i>¹³C 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 <i>δ</i>¹³C signal variation before leaf-level translocation of primary assimilates. The WUE_ge values in <i>P. orientalis</i> and <i>Q.  variabilis</i> 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 <i>P. orientalis</i> was significantly greater than that in <i>Q. variabilis</i>, 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 <i>δ</i>¹³C between water-soluble compounds (<i>δ</i>¹³C_WSC) and estimates based on gas-exchange observations (<i>δ</i>¹³C_obs) in <i>P. orientalis</i>, ranging between 0.0328 and 0.0472 ‰. Differences between <i>δ</i>¹³C_WSC and <i>δ</i>¹³C_obs in <i>Q. variabilis</i> increased as [CO₂] and SWC increased (0.0384–0.0466 ‰). The ¹³C fractionation from mesophyll conductance (<i>g</i>_m) and post-carboxylation both contributed to the total ¹³C fractionation that was determined by <i>δ</i>¹³C 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.