300M steel is an important material for fabricating main bearing structure of aircrafts. However, the surface oxidation and decarburization of this material will occur in varying degrees during the heat treatment of die forging process, which seriously affects the yield of forgings. Herein, the decarburization behavior of 300M steel under different heat treatment conditions is systematically studied. The morphology and thickness of the oxide layer are analyzed by scanning electron microscope (SEM) and the corresponding Energy Dispersive Spectrometer (EDS). The variation of decarburization depth is measured using microhardness method. The evolution of decarburization behavior of 300M steel under different heat treatment conditions is clarified, and the effect of anti-oxidation coating on the depth of decarburization layer is presented. The results show that the dense oxide layer formed on the surface of 300M steel during heat treatment has distinct protective effect, which can prevent the matrix carbon from diffusing to the surface, thus reduce the depth of decarburization layer. There is a balance between the diffusion rate of carbon and the surface oxidation rate at different temperatures, and the application of anti-oxidant coating on the surface of 300M steel will change the original balance. The results of microstructure analysis show that the full decarburization zone on the surface of 300M steel is composed by pearlite or ferrite, and the semi-decarburization zone is mainly martensite and precipitated carbide. We also expound the evolution of the near-surface microstructure of 300M steel with the increase of holding time. Finally, the coupling mechanism of decarburization and oxidation on the surface of 300M steel is revealed from the perspective of thermodynamics. The results of this paper are expected to provide important data and theoretical support for establishing optimization process of die forging and developing heat treatment protection technology of 300M steel.