For the induction of antigen-specific T-cell responses by vaccination, an appropriate immune adjuvant is required. Vaccine adjuvants generally provide two functions, namely, immune potentiator and delivery, and many adjuvants that can efficiently induce T-cell responses are known to have the combination of these two functions. In this study, we explored a cationic lipid DOTAP-based adjuvant. We found that the microfluidic preparation of DOTAP nanoparticles induced stronger CD4+ and CD8+ T-cell responses than liposomal DOTAP. The further addition of Type-A CpG D35 in DOTAP nanoparticles increased the induction of T-cell responses, particularly in CD4+ T cells. Further investigations revealed that the size of DOTAP nanoparticles, prepared buffer conditions, and physicochemical interaction with vaccine antigen are important factors for the efficient induction of T-cell responses with a relatively small antigen dose. These results suggested that microfluidicprepared DOTAP nanoparticles plus D35 are a promising adjuvant for a vaccine that induces therapeutic T-cell responses for treating cancer and infectious diseases.