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The global expansion of C-4 plants during the Late Cenozoic is an important event in the Earth’s environmental-ecosystem evolution. Taking into account the nonglobal expansion of C-4 plants, the CO2 concentration is not the only prerequisite for C-4 expansion. The direct driving factors for C-4 expansion may be associated with regional arid and/or warm seasonal precipitation and temperature variations. However, a large change in the CO2 concentration occurred, varying from 100 similar to 300 to > 2000 ppm during geological history; thus, it is necessary to understand the impact of such changes in the atmospheric CO2 concentration on the delta C-13 values of C-3 and C-4 plants during different geological periods. In this article, we analyzed the delta C-13 values for seven C-4 and C-3 plants grown under different CO2 concentrations for 10, 20, and 30 days. According to our study, the delta C-13 records for all the C-3 and C-4 plants show a rapid decrease followed by a slowly stable decreased trend with increasing CO2 concentration when the plants were grown for 10, 20, and 30 days. Comparing with the value averaging about -26 to -27 parts per thousand for C-3 plants and about -12 parts per thousand for C-4 plants, when the CO2 levels reach 20,000 ppm, all of the delta C-13 values of C-4 plants become more negative, varying from -27.5 to -36.1 parts per thousand, which fall into the range of delta C-13 values for C-3 plants. Namely, the increase of carbon dioxide can limit the identification of C-4 plants by carbon isotope composition. Thus, the expansion of C-4 plants as reconstructed from the geological records of carbon isotopes possibly does not reflect the origin and expansion of C-4 plants when atmospheric CO2 concentration was reduced to a threshold value, as previously thought. However, another possible explanation is that the atmospheric CO2 concentration was reduced to a threshold value from which the carbon isotope values of C-3 and C-4 plants can be distinguished. Therefore, we suggest that it is necessary to understand the delta C-13 range for C-3 and C-4 plants, to accurately evaluate the origin and expansion of C-4 plants, for geological periods when a high CO2 concentration occurred.