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Background: Heat shock proteins (HSPs) as an adjuvant induce antigen‐specific immunity through facilitating antigen presentation and stimulating T cells. In this study, the immunostimulatory properties of two major fragments of Hsp70 (N‐Hsp70(aa 1–387) with ATPase property and C‐Hsp70 (aa 508–641) with peptide‐binding capacity) and the full length of Hsp27 as vaccine adjuvants were evaluated to boost HIV‐1 Nef antigen‐specific immunity in both in vitro and in vivo experiments. Method s : At first, the nanoparticles harbouring DNA fusion constructs (i.e. N‐Hsp70‐Nef, C‐Hsp70‐Nef and Hsp27‐Nef) complexed with HIV Rev (34–50) cell‐penetrating peptide were generated to deliver DNA into the cells. Then, the recombinant Nef, Hsp27‐Nef, N‐Hsp70‐Nef and C‐Hsp70‐Nef proteins were generated in E.coli expression system. Next, the immunostimulatory properties of these fusion constructs were evaluated in both in vitro and in vivo studies. Finally, the secretion of main cytokines from single‐cycle replicable (SCR) HIV‐1 virion‐exposed splenocytes was investigated. Results: Our data showed that the stable and non‐toxic DNA/Rev nanoparticles could successfully deliver the genes of interest into the cells. Moreover, higher secretion of antibodies and cytokines was detected in mice receiving the Hsp‐Nef constructs than in mice receiving Nef antigen. The C‐Hsp70 was also superior for inducing Nef‐specific Th1 and CTL immunity compared with N‐Hsp70 and Hsp27. The T‐cell activity was maintained in the SCR‐exposed splenocytes, especially the splenocytes of mice receiving the C‐Hsp70‐Nef regimen. Conclusion: Altogether, these findings demonstrate the significance of Hsps as enhancers of antigen‐specific immunity. Notably, the C‐Hsp70 region showed better adjuvant properties for inducing cellular immunity in the improvement of HIV‐1 therapeutic vaccines. [ABSTRACT FROM AUTHOR]