Radioresistance poses the main challenge in radiation therapy (RT) for liver cancer, with the DNA Damage response (DDR) being a crucial component of this resistance. Ubiquitin-conjugating enzyme E2O (UBE2O) has been implicated in regulating tumor proliferation, cholesterol metabolism, and drug resistance. However, the role of the ubiquitin-conjugating enzyme E2O (UBE2O) in DDR of liver cancer remains to be fully explored. We discovered an elevated expression of UBE2O within liver cancer tissues, which was notably associated with unfavorable prognoses in hepatocellular carcinoma (HCC) patients. Furthermore, we found that the suppression of UBE2O can effectively reduce the growth and resistance to radiotherapy of HCC cells in vitro and in vivo. Moreover, p90 ribosomal S6 kinase2 (RSK2) was confirmed as a novel interacting kinase of UBE2O, which mediated the phosphorylation and degradation of UBE2O at the Thr838 site. RSK2 inhibition promotes tumor proliferation and resistance to radiotherapy of HCC cells in vitro and in vivo, and these effects are abrogated upon UBE2O knockdown. Collectively, our work revealed that UBE2O promotes tumor progression and resistance to radiotherapy, which was negatively regulated by RSK2 for phosphorylation and degradation, indicating that the RSK2/UBE2O axis provides a potential radiosensitization target for HCC patients.