Compared with the traditional monolayer culture, three-dimensional cell culture (TDCC) can more accurately simulate the growth environment of cells in vivo. In recent years, TDCC has been increasingly used in basic biological research. Epigenetic modification is closely linked to many cellular processes such as cell canceration. Radiation is one of the main means of tumor radiotherapy, the process of which is often accompanied by dynamic epigenetic changes, leading to DNA methylation, chromatin remodeling, histone modification and regulation of non-coding RNA. Exploring the mechanism of radiation-induced epigenetic regulation based on TDCC model is of great significance for treatment and protection of cancer patients.