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Root-zone CO₂ is essential for plant growth and metabolism. However, the partitioning and assimilation processes of CO₂ absorbed by roots remain unclear in various parts of the oriental melon. We investigated the time at which root-zone CO₂ enters the oriental melon root system, and its distribution in different parts of the plant, using ¹³C stable isotopic tracer experiments, as well as the effects of high root-zone CO₂ on leaf carbon assimilation-related enzyme activities and gene expressions under 0.2%, 0.5% and 1% root-zone CO₂ concentrations. The results showed that oriental melon roots could absorb CO₂ and transport it quickly to the stems and leaves. The distribution of ¹³C in roots, stems and leaves increased with an increase in the labeled root-zone CO₂ concentration, and the δ¹³C values in roots, stems and leaves increased initially, and then decreased with an increase in feeding time, reaching a peak at 24 h after ¹³C isotope labeling. The total accumulation of ¹³C in plants under the 0.5% and 1% ¹³CO₂ concentrations was lower than that in the 0.2% ¹³CO₂ treatment. However, the distributional proportion of ¹³C in leaves under 0.5% and 1% ¹³CO₂ was significantly higher than that under the 0.2% CO₂ concentration. Photosynthetic carbon assimilation-related enzyme activities and gene expressions in the leaves of oriental melon seedlings were inhibited after 9 days of high root-zone CO₂ treatment. According to these results, oriental melon plants’ carbon distribution was affected by long-term high root-zone CO₂, and reduced the carbon assimilation ability of the leaves. These findings provide a basis for the further quantification of the contribution of root-zone CO₂ to plant communities in natural field conditions.