Background and Aims Liver cancer is a common malignant tumor of the digestive system. Tumor recurrence is a major cause of treatment failure. High mobility group box 1 (HMGB1) has been found to be highly expressed in a variety of cancers, and play an important role in cancer development and progression. Therefore, this study was designed to investigate the relations of HMGB1 with the repopulation of liver cancer cells and the prognosis of liver cancer through in vitro repopulation model of liver cancer cells and database analysis.Methods Liver cancer Huh7 and Li7 cells were chosen for this study. The two types of cells were transfected with plasmids carrying firefly luciferin and green fluorescent protein (Fluc-GFP) gene to construct respective reporter cells Huh7-Fluc and Li7-Fluc cells. The Huh7 and Li7 cells treated with X-ray irradiation at a dose of 10 Gy were used as feeder cells and then co-cultured with their corresponding reporter cells to construct the in vitro repopulation models, using the systems of pure reporter cells and co-culture of the reporter cells and feeder cells without X-ray treatment as controls. The growth conditions of the reporter cells were evaluated by analyzing the changes in their luciferase activities via bioimaging, on which the effects of HMGB1 inhibitor glycyrrhizin (Gly) intervention were also observed. The HMGB1 expression levels of liver cancer tissue and tumor adjacent tissue as well as its association with the prognosis of liver cancer were analyzed using the TIMER2.0 and GEPIA2 tumor data analysis platforms.Results Compared with respective pure reporter cells group and reporter cells plus non-irradiated feeder cells co-culture group, the growth abilities of the reporter cells in both repopulation groups were significantly enhanced (all P<0.01). After Gly intervention, the growth abilities of the reporter cells in both repopulation models were significantly suppressed (both P<0.01), but the growth conditions of the pure reporter cells showed no significant changes (both P>0.05). The results of public database retrieval showed that the expression level of HMGB1 was higher in liver cancer than that in adjacent tissue (P<0.01), and the overall survival time of liver cancer patients with high HMGB1 expression level was significantly shorter than those with low HMGB1 expression level (P<0.01), and HMGB1 expression level was not correlated with disease free survival of liver cancer patients (P>0.05).Conclusion HMGB1 is involved in liver cancer cell repopulation induced by X-ray irradiation. The HMGB1 expression level in liver cancer tissue can be used as reference index for predicting the prognosis of overall survival of liver cancer patients, which may also provide a new strategy for the clinical treatment of liver cancer.