Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world and is associated with high mortality. Ionizing radiation (IR)-based therapy causes DNA damage, exerting a curative effect; however, DNA damage repair signaling pathways lead to HCC resistance to IR-based therapy. RAD21 is a component of the cohesion complex, crucial for chromosome segregation and DNA damage repair, while it is still unclear whether RAD21 is implicated in DNA damage and influences IR sensitivity in HCC. The current research explores the effect and upstream regulatory mechanism of RAD21 on IR sensitivity in HCC. In the present study, RAD21 mRNA and protein expression were increased within HCC tissue samples, particularly within IR-insensitive HCC tissues. The overexpression of RAD21 partially attenuated the roles of IR in HCC by promoting the viability and suppressing the apoptosis of HCC cells. RAD21 overexpression reduced the culture medium 8-hydroxy-2-deoxyguanosine concentration and decreased the protein levels of γH2AX and ATM, suggesting that RAD21 overexpression attenuated IR treatment-induced DNA damage to HCC cells. miR-320b targeted RAD21 3'-UTR to inhibit RAD21 expression. In HCC tissues, particularly in IR-insensitive HCC tissues, miR-320b expression was significantly downregulated. miR-320b inhibition also attenuated IR treatment-induced DNA damage to HCC cells; more importantly, RAD21 silencing significantly attenuated the effects of miR-320b inhibition on IR treatment-induced DNA damage, suggesting that miR-320b plays a role through targeting RAD21. In conclusion, an miR-320b/RAD21 axis modulating HCC sensitivity to IR treatment through acting on IR-induced DNA damage was demonstrated. The miR-320b/RAD21 axis could be a novel therapeutic target for further study of HCC sensitivity to IR treatment.