Aim: To study the potential chemopreventive effect of dimethyl dicarboxylate biphenyl (DDB), an anti-hepatitis drug, on hepatocarcinogenesis in vitro.Methods: The anti-carcinogenesis effect of DDB was assessed on a two-stage chemical oncogenesis model induced by 3-methylcholanthrene and 12-O-tetradecanoyl phorbol 13-acetate (TPA) with WB-F344 rat liver epithelial cells(WB-F344 cells) in vitro. A soft-agar colony formation assay was used to determine the tumorigenic potential of the transformed WB-F344 cells. The gap junctional intercellular communication (GJIC) was detected using the scrape loading/dye transfer technique. Results: DDB at 1 μmol/L, 2 μmol/L, and 4 μmol/L significantly prevented the malignant transformation of WB-F344 cells induced by 3-methylcholanthrene and TPA. The average number of transformed foci decreased dramatically by 10.0%, 37.2%, and 47.4%, respectively. In soft agar, a remarkable decrease in colony numbers was observed in transformed cells treated with 2 μmol/L and 4 μmol/L DDB. DDB at 1 μmol/L, 2 μmol/L, and 4 μmol/L inhibited the downregulation of GJIC induced by TPA in a dose-dependent manner. The GJIC recovered to 25.6%, 34.6%, and 44.9%, respectively, of the control WB-F344 cells by DDB. Conclusion: DDB has a potential chemopreventive effect on hepatocarcinogenesis induced by carcinogens in vitro.