The development of multidrug resistance (MDR) in hepatocellular carcinoma (HCC) may markedly reduce the efficacy of its chemotherapeutic treatment. However, the mechanism regulating the development of MDR in these tumors remains unknown. Given the emerging role of small ubiquitin‑like modifier (SUMO)ylation in tumorigenesis, the possibility that it may also be involved in MDR development was investigated. The expression of SUMO‑1 was first analyzed using immunohistochemistry in 20 cases of HCC. Nuclear SUMO‑1 immunostaining was observed to be significantly increased in HCC specimens compared with matched adjacent non‑neoplastic controls. To further investigate the potential role of SUMOylation in MDR in HCC, a multidrug‑resistant HCC cell line, HepG2/R, was established by exposing HCC cells to gradually increasing concentrations of 5‑fluorouracil. Western blot analysis revealed that the total levels of SUMO‑1‑conjugated proteins were markedly increased in HepG2/R cells compared with parental HepG2 cells. Furthermore, the expression of ubiquitin‑like modifier activating enzyme 2 and sentrin‑specific protease 1, important enzymes in the SUMOylation cascade, were markedly upregulated in the HepG2/R cell line. These findings support the hypothesis that SUMOylation is important in the development of MDR in HCC.