Atmospheric pCO₂ concentrations have been estimated for intervals of the Eocene using various models and proxy information. Here we reconstruct late middle Eocene (42.0–38.5 Ma) pCO₂ based on the fossil leaves of Nageia maomingensis Jin et Liu collected from the Maoming Basin, Guangdong Province, China. We first determine relationships between atmospheric pCO₂ concentrations, stomatal density (SD) and stomatal index (SI) using “modern” leaves of N. motleyi (Parl.) De Laub, the nearest living species to the Eocene fossils. This work indicates that the SD inversely responds to pCO₂, while SI has almost no relationship with pCO₂. Eocene pCO₂ concentrations can be reconstructed based on a regression approach and the stomatal ratio method by using the SD. The first approach gives a pCO₂ of 351.9 ± 6.6 ppmv, whereas the one based on stomatal ratio gives a pCO₂ of 537.5 ± 56.5 ppmv. Here, we explored the potential of N. maomingensis in pCO₂ reconstruction and obtained different results according to different methods, providing a new insight for the reconstruction of paleoclimate and paleoenvironment in conifers.