Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality, characterized by a high rate of postoperative recurrence and poor long-term survival outcomes. Structural maintenance of chromosome 4 (SMC4) is frequently overexpressed in various types of cancer and plays a pivotal role in tumor cell growth, migration, and invasion. Bioinformatics analysis has revealed a significant correlation between the tumor-node metastasis (TNM) stage (P <
0.01) and SMC4 expression (P <
0.05), and SMC4 was associated with poor prognosis in HCC. Furthermore, SMC4 was identified as an independent prognostic factor for HCC. Ubiquitin-specific peptidase 39 (USP39) was found whether the regulation was observed to affect protein synthesis or stability through bioinformatics analysis and immunoprecipitation. The expression levels and cellular localization of SMC4 and USP39 in hepatoma cells were evaluated using quantitative real-time PCR (qPCR), western blotting, and immunohistochemistry (IHC), all of which indicated significantly elevated expression of USP39 and SMC4 in HCC. The roles of the SMC4/USP39 were further investigated through several assays, including the 3-(4,5-Dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and wound healing assay. The results demonstrated that USP39/SMC4 plays a crucial role in enhancing the viability and proliferation of HepG2 cells. Additionally, bioinformatics analysis identified ZNF207 and TIAL1 as potential target proteins of SMC4. Drug-resistant hepatoma cell lines were established, and both MTT and EdU assays were performed to assess cell viability and proliferation. The results demonstrated that HepG2/5-FU cells regained their sensitivity to 5-FU following the knockdown of SMC4. Additionally, the knockdown of either TIAL1 or ZNF207 also restored 5-FU sensitivity in HepG2/5-FU cells, effectively inhibiting cell viability and proliferation. Our study underscores the significant role of the USP39/SMC4 in HCC development and suggests that SMC4 may contribute to the regulation of drug resistance in hepatoma cell lines, potentially through interactions with TIAL1 and ZNF207.