OBJECTIVE: To investigate the effect of hepatitis B virus (HBV) and hepatitis B virus surface antigen (HBsAg) on microRNA-31 (miR-31) expression in HBV-related hepatocellular carcinoma using HepG2 hepatoma cells. METHODS: Stable HBsAg-overexpressing cell lines were established by transfecting HepG2 cells with an HBsAg-bearing mammalian expression vector, and the clones (designated as HepG2-H2 cells) were validated by enzyme-linked immunosorbent assay and immunohistochemistry. Effects on expression of miR-31 were determined by measuring the mRNA level by real-time PCR and performing statistical comparisons with levels detected in HepG2-H0 cells (stably transfected with empty vector) and HepG2.2.15 cells (stably transfected with the HBV genome). RESULTS: The HepG2-H2 HBsAg-overexpressing transfectant cell line was successfully established. The expression level of miR-31 was significantly higher in the HepG2-H0 cells than in the HepG2.2.15 cells (t = 583.8, P less than 0.001). In contrast, the expression level of miR-31 was significantly higher in the HBsAg-overexpressing HepG2-H2 cells than in the HepG2-H0 cells (F = 24.9, P less than 0.05). CONCLUSION: Intact HBV leads to down-regulation of miR-31 expression and HBsAg overexpression leads to up-regulation of miR-31 expression in hepatocarcinoma cells.