Yersinia Pestis outer proteins, plasminogen activator protease and Yop secretion protein F are necessary for the full virulence of Yesinia pestis and have been proposed as potential protective antigens for vaccines against plague. In the current study, we used DNA immunization as a tool to study the relative protective immunity of these proteins with a standardized intranasal challenge system in mice. While the natural full-length gene sequences for most of these Y. pestis proteins did not display a good level of protein expression in vitro when delivered by a DNA vaccine vector, the overall immunogenicity of these wild type gene DNA vaccines was low in eliciting antigen-specific antibody responses and gene sequence modifications improved both of these parameters. However, even modified YopD, YopO and YscF antigens were only able to partially protect immunized mice at various levels against lethal challenge with Y. pestis KIM 1001 strain while no protection was observed with either the YopB or Pla antigens. These results demonstrate that DNA immunization is effective in screening, optimizing and comparing optimal antigen designs and immunogenicity of candidate antigens for the development of a subunit-based plague vaccine.