DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. In this study, we report that coadministration of a hepatitis B virus (HBV) DNA vaccine with prothymosin alpha as an adjuvant improves antibody responses to HBV S antigen. We also observed higher seroconversion rates and higher antibody titers. Prothymosin alpha appears to increase the number and affinity of hepatitis B surface antigen-specific, gamma interferon-secreting T cells and to enhance cellular immune response to the PreS2S DNA vaccine. Interestingly, administering the DNA separately from the prothymosin alpha plasmid abrogated the enhancement of DNA vaccine potency. The results suggest that prothymosin alpha may be a promising adjuvant for DNA vaccines.