The change in ancient atmospheric CO₂ concentrations provides important clues for understanding the relationship between the atmospheric CO₂ concentration and global temperature. However, the lack of CO₂ evolution curves estimated from a single terrestrial proxy prevents the understanding of climatic and environmental impacts due to variations in data. Thus, based on the stomatal index of fossilized Metasequoia needles, we reconstructed a history of atmospheric CO₂ concentrations from middle Miocene to late Early Pleistocene when the climate changed dramatically. According to this research, atmospheric CO₂ concentration was stabile around 330–350 ppmv in the middle and late Miocene, then it decreased to 278–284 ppmv during the Late Pliocene and to 277–279 ppmv during the Early Pleistocene, which was almost the same range as in preindustrial time. According to former research, this is a time when global temperature decreased sharply. Our results also indicated that from middle Miocene to Pleistocene, global CO₂ level decreased by more than 50 ppmv, which may suggest that CO₂ decrease and temperature decrease are coupled.