Three-dimensional cell culture is a novel type of in vitro culture method.This system can mimic the microenvironment of cell growth in vivo,where cells exhibit similar state and function to that in the in vivo environment.Currently,three-dimensional culture system has been widely applied in tissue engineering and tumor cell biology fields,etc.Radiotherapy is an important treatment of cancer.Radioresistance of tumor cells is the main cause of treatment failure,tumor recurrence and metastasis.Tumor cells can exhibit the resistant characteristics in the three-dimensional culture system,similar to those of tumor cells in vivo.Therefore,three-dimensional culture system can be adopted to investigate the radioresistance and underlying mechanism of tumor cells and identify the key factors regulating the radioresistance of tumor cells,which plays a pivotal role in enhancing the radioresistance and improving the effect of radiotherapy.This article will review recent research progress in the radioresistance and sensitization of tumor cells under three-dimensional culture system,aiming to provide reference for relevant investigations.