Background Treatment of HBV chronic carriers using interferon (IFN)-a or nucleoside/nucleotide analogues fails to suppress viral infection. Type-II IFN-? has been shown to inhibit HBV replication. The goal of the present work was to evaluate the antiviral efficacy against HBV of a thermostable IFN-? variant isolated using Massive Mutagenesis® and thermoresistant selection (THR™) technologies.Methods The thermostability of wild-type (wt) and S63C IFN-? was determined in vitroand in vivo. Activation of the IFN-? responsive element by wt and S63C IFN-? was tested using a luciferase assay. HepG2.2.15 cells constitutively expressing HBV were used to analyse the antiviral activity of wt and S63C IFN-? against HBV replication. Intracellular HBV DNA was detected by Southern blot and quantified by real-time PCR analyses.Results S63C IFN-? was shown to be more thermostable and had a longer half-life than wt IFN-?. Both wt and S63C IFN-? displayed a similar capacity to activate the IFN pathway. The treatment of HepG2.2.15 cells with wt or S63C IFN-? induced the inhibition of HBV viral replication. After heating, S63C IFN-? displayed better conservation of its antiviral activity against HBV when compared with wt IFN-?.Conclusions These results confirm that the THR™ method can be used to isolate mutants with enhanced thermostability and demonstrate that a thermostable IFN-? variant presents antiviral properties against HBV replication. This molecule could provide a new strategy to treat patients who do not respond to antiviral therapy.