Effective antibody responses provide crucial immunity against influenza (Flu) virus infection. The hemagglutinin (HA) protein is the major target of protective antibody responses induced by viral infection and by vaccination with both inactivated and live attenuated Flu vaccines but the knowledge on the optimal designs of protective HA antigens from different Flu serotypes is still limited. In this study, we have significantly improved the immunogenicity of HA expressing DNA vaccines by using codon optimized HA sequences for either an H1 serotype (A/NewCal/20/99) or an H3 serotype (A/Panama/2007/99) human influenza A virus, and then used these constructs as model antigens to identify the optimal HA antigen designs to elicit high level protective antibody responses. Two forms of HA antigen, a wild type full length HA and a secreted form with transmembrane (TM) domain truncated HA, were produced. Both forms of HA DNA vaccines, either from H1 or H3 serotypes, were able to elicit high levels of HA-specific IgG responses, in immunized rabbits as measured by ELISA. Interestingly, their abilities in eliciting hemagglutination inhibition (HI) and neutralizing antibody (NAb) responses differ. For the H1 HA antigens, the full length HA induced significantly higher HI and NAb responses than the TM truncated HA. For the H3 HA antigen, both the full length HA and TM truncated HA induced high levels of HI and NAb responses. These data indicate that H1 and H3 antigens have different expression requirements for the induction of an optimal protective antibody response and that the structure integrity of HA antigens is critical in eliciting type specific protective antibody responses. Our findings would have an important impact to future subunit based Flu vaccine development.