Interleukin-21 (IL-21) is a T-cell-derived cytokine that modulates T-cell, B-cell, and natural killer cell responses. It is not known if it could be used as an adjuvant for HIV DNA vaccination. In our study, we investigated if a DNA construct expressing IL-21 (designated as pVAX-IL-21) as a molecule adjuvant could enhance antigen-specific immune responses to an HIV DNA vaccine (pGX-EnvC). We found that a higher level of antigen-specific cytotoxic responses was induced in BALB/C mice immunized with pGX-EnvC with the pVAX-IL-21 via electroporation. The increased response was associated with higher expression of IFN-γ in CD8⁺ T cells. In contrast, the administration of pVAX-IL-21 inhibited the antibody responses to HIV induced by the pGX-EnvC. The plasma cell inhibitory transcription factors B-cell lymphoma 6 protein (Bcl-6) and Pax-5 were increased in B cells from mice that had been immunized by HIV DNA vaccine plus pVAX-IL-21, suggesting that the expressed IL-21 may inhibit the differentiation from B cells to plasma cells. These results indicate that IL-21 could enhance CD8⁺ T-cell immunity, but inhibit humoral responses during HIV DNA vaccination.