Abstract: Objective To investigate the effect of tripartite motif-containing protein 31 (Trim31) on sorafenib resistance in hepatocellular carcinoma (HCC) cells and the underlying mechanisms. Methods The GEPIA database was used to analyze the levels of Trim31 mRNA in HCC. The protein levels of Trim31 in HCC tissues were detected by Western blot. A Trim31-overexpressing HCC cell line was constructed, and the effect of Trim31 overexpression on HCC cell proliferation were assessed by CCK-8, colony formation, and EdU assays. Protein enrichment analysis and Western blot were used to screen and validate the downstream signaling pathways of Trim31. Furthermore, Trim31-targeting shRNA was screened, and a stable Trim31-silenced HCC cell line was established. The effect of Trim31 silencing on HCC cell proliferation was evaluated by Western blot, and CCK-8, colony formation, and EdU assays. Moreover, the effect of P38 overexpression on HCC cell proliferation in the context of Trim31 overexpression was observed to elucidate the regulatory relationship between Trim31 and the P38 pathway. Sorafenib-treated Trim31-overexpressing HCC cell lines were used to assess the impact of Trim31 overexpression on HCC cell proliferation post-sorafenib treatment through CCK-8, colony formation, EdU assays, and Western blot. A sorafenib-resistant HCC cell line was established, where Trim31 was silenced in this cell line. The effect of Trim31 silencing on the proliferation of the sorafenib-resistant cell line was evaluated by CCK-8, colony formation, EdU assays, and Western blot. Results Trim31 mRNA was highly expressed in HCC tissues. Trim31 overexpression promoted HCC cell proliferation, while Trim31 silencing inhibited it. P38 overexpression suppressed the proliferative effect of Trim31 overexpression in HCC cells, suggesting a possible negative regulatory relationship between Trim31 and the P38 pathway. Trim31 overexpression attenuated the inhibitory effect of sorafenib on HCC cell proliferation. Although sorafenib did not affect the proliferation of the resistant HCC cell line, Trim31 silencing significantly inhibited the proliferation of the sorafenib-resistant HCC cells. Conclusions Trim31 mRNA is highly expressed in HCC, and silencing Trim31 can reduce the resistance of HCC cells to sorafenib, potentially through the activation of the P38 pathway.