Development of an effective vaccine against HIV-1 infection is a main concern in worldwide. A potent vaccine for HIV-1 requires the induction and maintenance of both humoral and cellular immunity. In this study, the levels of humoral and cellular immune responses were compared using MPER-V3 injection in three immunization strategies such as DNA/DNA, peptide/peptide, and DNA/peptide (prime-boost). MPG peptide and Montanide 720 were used as a DNA delivery system, and as a peptide adjuvant, respectively. Our results demonstrated that MPG forms stable non-covalent nanoparticles with plasmid DNA at N/P ratio of 10:1 (∼ 110-130 nm). The in vitro transfection efficiency of MPER-V3 DNA using MPG was comparable with lipofectamine and turbofect reagents as a common delivery system. In vivo prime-boost immunization using HIV-1 MPER-V3 could significantly enhance humoral and cellular immune responses as compared to control groups. The mixture of IgG1 and IgG2a was observed for each strategy, but IFN-γ production was significantly higher in prime-boost and peptide immunizations than that in DNA immunizations, inducing Th1 response. Moreover, our data showed that prime immunization with low dose of the nanoparticles (MPER-V3 DNA: MPG at ratio of 1:10) followed by MPER-V3 peptide drives T cell responses towards a Th1-type similar to high dose of the naked DNA prime/peptide boost immunization. Generally, the prime-boost strategy could improve both immune responses against MPER and especially V3 peptides suggesting its application as a promising HIV vaccine candidate in future.