Background: High mobility group box 1 (HMGB1) is a highly conserved protein present in the nuclei and cytoplasm of cells which has an important role as a mediator of inflammation in the extracellular environment. HMGB1 was identified as an innate adjuvant that induces immune responses against soluble antigens in vivo.Objective: Our goal is the generation of recombinant HMGB1-GFP fusion protein in insect cells for evaluation of immune responses in mouse model. Method: In the current study, we used a baculovirus expression system for insect cells that was based on expression of HMGB1 with target gene (GFP), and purified the recombinant HMGB1- GFP fusion protein. We then demonstrated whether immunogenicity of GFP changes in the presence or absence of recombinant HMGB1 acting as an adjuvant in C57BL/6 and BALB/c mice. Results: Our data showed that HMGB1 had a major influence on antibody immune responses induced by GFP in both animal models. The groups receiving HMGB1-GFP fusion protein showed total IgG and IgG2a responses significantly higher than IgG1 in BALB/c mice. Indeed, a mixed IgG1/IgG2a response was observed with high intensity toward IgG2a. In contrast, C57BL/6 mice immunized by HMGB1-GFP protein elicited the same levels of IgG1 and IgG2a. However, the levels of IgG2a and total IgG against the recombinant GFP (rGFP) in C57BL/6 mice were lower than those in BALB/c mice. Conclusion: We concluded that fusion of HMGB1 with GFP was immunologically more effective than GFP alone.