With the increasing challenges of climate change caused by global warming, the effective reduction of carbon dioxide (CO₂) becomes an urgent environmental issue for the sustainable development of human society. Previous reports indicated increased biomass in genetically engineered (GE) Arabidopsis and rice overexpressing the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, suggesting the possibility of consuming more carbon by GE plants. However, whether overexpressing the EPSPS gene in GE plants consumes more CO₂ remains a question. To address this question, we measured expression of the EPSPS gene, intercellular CO₂ concentration, photosynthetic ratios, and gene expression (RNA-seq and RT-qPCR) in GE (overexpression) and non-GE (normal expression) Arabidopsis and rice plants. Results showed substantially increased EPSPS expression accompanied with CO₂ consumption in the GE Arabidopsis and rice plants. Furthermore, overexpressing the EPSPS gene affected carbon-fixation related biological pathways. We also confirmed significant upregulation of four key carbon-fixation associated genes, in addition to increased photosynthetic ratios, in all GE plants. Our finding of significantly enhanced carbon fixation in GE plants overexpressing the EPSPS transgene provides a novel strategy to reduce global CO₂ for carbon neutrality by genetic engineering of plant species, in addition to increased plant production by enhanced photosynthesis.