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Tomatoes are a perishable and seasonal fruit with a high economic impact. Carbon dioxide (CO₂), among several other reagents, is used to extend the shelf-life and preserve the quality of tomatoes during refrigeration or packaging. To obtain insight into CO₂ stress during tomato ripening, tomatoes at the late green mature stage were conditioned with one of two CO₂ delivery methods: 5% CO₂ for 14 days (T1) or 100% CO₂ for 3 h (T2). Conventional physical and chemical characterization found that CO₂ induced by either T1 or T2 delayed tomato ripening in terms of color change, firmness, and carbohydrate dissolution. However, T1 had longer-lasting effects. Furthermore, ethylene production was suppressed by CO₂ in T1, and promoted in T2. These physical observations were further evaluated via RNA-Seq analysis at the whole-genome level, including genes involved in ethylene synthesis, signal transduction, and carotenoid biosynthesis. Transcriptomics analysis revealed that the introduction of CO₂ via the T1 method downregulated genes related to fruit ripening; in contrast, T2 upregulated the gene encoding for ACS6, the enzyme responsible for S1 ethylene synthesis, even though there was a large amount of ethylene present, indicating that T1 and T2 regulate tomato ripening via different mechanisms. Quantitative real-time PCR assays (qRT-PCR) were used for validation, which substantiated the RNA-Seq data. The results of the present research provide insight into gene regulation by CO₂ during tomato ripening at the whole-genome level.