Background:　A　therapeutic　vaccine　for　chronic　hepatitis　B　virus　(HBV)　infection　that　enhances　virus-specific　cellular　immune　responses　is　urgently　needed.　The　＂prime-boost＂　regimen　is　a　widely　used　vaccine　strategy　against　many　persistence　infections.　However,　few　reports　have　addressed　this　strategy　applying　for　HBV　therapeutic　vaccine　development.　Methodology/Principal　Findings:　To　develop　an　effective　HBV　therapeutic　vaccine,　we　constructed　a　recombinant　vaccinia　virus　(Tiantan)　containing　the　S+PreS1　fusion　antigen　(RVJSS1)　combined　with　the　HBV　particle-like　subunit　vaccine　HBVSS1　to　explore　the　most　effective　prime-boost　regimen　against　HBV.　The　immune　responses　to　different　prime-boost　regimens　were　assessed　in　C57BL/C　mice　by　ELISA,　ELISpot　assay　and　Intracellular　cytokine　staining　analysis.　Among　the　combinations　tested,　an　HBV　protein　particle　vaccine　priming　and　recombinant　vaccinia　virus　boosting　strategy　accelerated　specific　seroconversion　and　produced　high　antibody　(anti-PreS1,　anti-S　antibody)　titres　as　well　as　the　strongest　multi-antigen　(PreS1,　and　S)-specific　cellular　immune　response.　HBSS1　protein　prime/RVJSS1　boost　immunization　was　also　generated　more　significant　level　of　both　CD4+　and　CD8+　T　cell　responses　for　Th1　cytokines　(TNF-alpha　and　IFN-gamma).　Conclusions:　The　HBSS1　protein-vaccine　prime　plus　RVJSS1　vector　boost　elicits　specific　antibody　as　well　as　CD4　and　CD8　cells　secreting　Th1-like　cytokines,　and　these　immune　responses　may　be　important　parameters　for　the　future　HBV　therapeutic　vaccines.