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The greatest biomedical challenge of this century is to develop a preventive vaccine against Human Immunodeficiency Virus (HIV-1). For an HIV vaccine to be effective, it appears logical to develop new strategies that enhance the level of the immune response as well as steer it towards the desirable cellular type. In view of this, there is a need for rational inclusion of biological adjuvants into the HIV-1 vaccination strategies that could potentiate the immune responses both qualitatively and quantitatively. The adjuvant may include the immunostimulatory oligonucleotides containing CpG motifs, whose immunomodulatory characters are well established and represent the basis for an effective vaccine adjuvant. In our study, we investigated the use of an immunostimulatory oligonucleotide (or CpG motif), 1826-ODN to augment the immune response elicited by plasmid DNA vaccine constructs containing Indian subtype C HIV-1 envelope gp120 and gag-protease genes in Balb/c mouse model system. A dose of 2-µg CpG motifs/mouse was found to be optimum when co-administered with the DNA vaccine constructs with the peak level of humoral and cell mediated immune responses at 6 weeks post immunization. Murine IFN-γ ELISpot assay demonstrated that the use of 1826-ODN led to a broad based and long term recognition of the subtype C envelope and gag peptides. The use of CpG motifs has been effective in augmenting the immune responses generated by the DNA constructs. Taken together, these results are an important advancement towards the design of future preclinical and clinical trials of these vaccine constructs.