Objective:To investegate whether codon optimization of middle hepatitis B surface antigen could enhance the immunogenicity of HBV DNA vaccine. Methods:According to the sequence of amino acids of MHBs(adr subtype), the codon optimized genes were designed and synthesized without changing the sequence of amino acids and then cloned into vector pSW3891, which was named pSW3891/MHBs/adr/opt. 293T cells were transiently transfected with opt and wild-type MHBs DNA vaccine(pSW3891/MHBs/adr) and empty vector pSW3891. The protein expression level was assessed by western blot. BALB/c mice were intramuscularly injected with opt, adr or pSW3891. Anti-HBs in sera was tested by ELISA. IFN-γsecretion splenocytes of mice immunized with opt, adr and pSW3891 were tested by ELISPOT. Results:The level of protein expression in both supernatant and lysate of 293T cells transfected with opt is higher than that in 293T cells transfected with adr. The BALB/c mice immunized with opt showed stronger anti-HBs response and more IFN-γsecretion splenocytes than those immunized with adr. Conclusion:Codon optimization of MHBs DNA vaccine could improve protein expression and induce significant humoral and cellular immune responses in BALB/c mice.