In vitro three-dimensional cell culture (TDCC) is a new cell culture model that owns its unique perspectives by mimicking features of the in vivo environment and is believed to have the potential to bridge the gap between traditional cell culture and animal models in biological and medicinal studies. Despite the generally harmful and potentially lethal effects, ionizing radiation also demonstrates health benefits in radiotherapy for cancers. This review mainly introduced the three-dimensional cell culture model and different biological effects induced by ionizing radiation, as well as their potential mechanism(s) as compared to traditional monolayer cultures.