Pre-oxidation is one of the key steps in the preparation of carbon fibers, and most of the defects generated in this process are inherited by the carbon fibers. Therefore, the structural optimization of polyacrylonitrile (PAN) pre-oxidized fibers is particularly important for enhancing the mechanical properties of carbon fibers. Irradiation is a modification method for optimizing the structure of pre-oxidized PAN fibers and effectively improving their mechanical properties. In this study, the microstructural evolution of high-dose electron beam irradiated PAN fibers during the pre-oxidation is investigated. Synchrotron radiation in situsmall angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) were used to characterize the heat treatment process of high-dose irradiated and unirradiated PAN fibers. Additionally, the characterization of PAN pre-oxidized fibers was analytically investigated by combining differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FTIR).The results showed that high-dose irradiation (500 kGy) promoted the cross-linking and cyclization of the PAN fibers during heat treatment, thus effectively alleviating the intense exotherms caused by the cyclization and reducing the initial cyclization temperature. Additionally, high-dose irradiation significantly affected the molecular structure of the PAN fibers, whereas high-dose irradiation of the PAN fibers during the heat treatment reduced the transverse microporous (D) size, orientation angle (B_eq), and crystal-layer spacing d₀₀₂ to 7.61 nm, 8.58°, and 0.352 nm, respectively. In summary, high-dose irradiation improves the microstructure of the PAN fibers after heat treatment, which facilitates improvements to the structure and the properties of the corresponding carbon fibers.