DNA vaccination that can induce both cellular and humoral immune response has become an attractive immunization strategy against cancer and infectious disease. Elucidation of the precise mechanisms of immune priming will be important in the development of effective DNA vaccines. In this review, we illustrate possible mechanisms in priming cytotoxic T cell response involving the intracellular degradation, processing and presentation of encoded antigen. We also discuss the roles of costimulatory molecules expressed on antigen-presenting cells (APCs) in inducing optimal CTL activity. Hence, a rational strategy for increasing DNA potency would be to facilitate these pathways. Additionally, we focus on recent strategies including rapid degradation of ubiquitin-antigen fusion proteins, direct targeting to APCs for increased DNA uptake, direct routing an antigen into the MHC class I and II processing and presentation pathways, and increasing the immunogenicity of encoded antigen. All of these approaches have resulted in increased potency of DNA vaccines.