Biphenyl dimethyl dicarboxylate (DDB) is a hepatoprotectant, which is used as an adjuvant agent in a treatment for chronic hepatitis. Amantadine is an antiviral agent, which is utilized primarily in the treatment of influenza, but also, occasionally in the treatment of hepatitis C. In a previous study, we reported that DDB, coupled with amantadine, would exert an anti-HBV effect, via the induction of interferon-inducible gene expression in the HepG2 2.2.15 cell line. The primary objective of the present study was to determine whether or not DDB and/or amantadine exhibit anti-HBV properties, and what mechanisms of action might be involved in such properties. In our study, we were able to determine that DDB stimulates Jak/Stat signaling, and induces the expression of interferon alpha (IFN-α) stimulated genes, most notably 6-16 and ISG12. In addition, the antiviral effectors induced by IFN-α, PKR, OAS, and MxA, were regulated in the presence of DDB at its optimal concentration (250 μg/mL), to a degree commensurate with the degree of induction associated with the IFN-α treated group. Finally, we determined that the replication of pregenomic RNA and HBeAg was inhibited by DDB treatment, and this inhibition was maximized when coupled with the administration of amantadine (25 μg/mL). In conclusion, the results of this study demonstrated clearly that DDB, as well as the combination of DDB/amantadine, directly inhibited IFN-α signaling-mediated replication of HBV in infected hepatocytes, and thus may represent a novel treatment for chronic hepatitis B, which would be characterized principally by its improved safety over other treatment strategies.