Abstract: Objective To investigate the effect of FAT10 regulating autophagy in hepatocellular carcinoma (HCC) cells and determine the function of autophagy in the radiosensitivity of HCC cells. Methods The regulating effect of FAT10 on autophagy-related proteins in HCC cells and clinical specimens were detected by Western blot and immunohistochemistry (IHC). Clonogenic assay was employed to observe the changes of radiosensitivity in HCC cells under a regulation of autophagy. Results Elevated FAT10 expression was associated with significant increase of autophagy-related proteins Beclin1and LC3-Ⅰ/Ⅱ and decrease of p62 proteins in HCC cells, especially when underwent an X-ray irradiation. A significant correlation between FAT10 and Beclin1 expression was further conformed by IHC analysis from 35 HCC specimens (P<0.01), and FAT10 overexpression was mostly accompanied by elevated Beclin1 expression. Furthermore, the increased expression of FAT10 was associated with dramatical radiation resistance, and knockdown of FAT10 displayed significantly radiosensitivity. However, when treated with 3-MA, a cell-permeable autophagic sequestration blocker, or specifical knockdown of Beclin1, there was no obvious changes in the radiosensitivity of HCC cells when FAT10 expression was changed. Conclusion Overexpression of FAT10 is associated with an enhancement of autophagy and enhances the radiosensitivity of HCC cells.