Objective To explore the influence of adenovirus carrying Galectin9 gene on the survival of cardiac transplant and the differentiation and proliferation of CD4+CD25+Treg cells in mice receiving heterotopic heart transplantation.Methods Neck heterotopic heart transplantation models were established with male BALB/c→C57BL/6 mice by Cuff method, and they were randomly divided into 3 groups (n=10): control group (received 0.9% sodium chloride solution after operation), adenovirus group, and pAdeasyTM Galectin9 treatment group. The survival time of the graft hearts was recorded. The spleens of mice were collected when the graft hearts stopped beating or 14 days after operation; the mononuclear cells were isolated and the proportion of CD4+CD25+ Treg cells was determined by flow cytometry. Treg cell related cytokines (IL10, TGFβ1) in the peripheral blood were assayed by ELISA.Results The cardiac allografts in pAdeasyTM Galectin9 group survived significantly longer than those in the other two groups (P<0.01), and the proportion of CD4+CD25+ Treg cells in CD4 + T cells was significantly higher(P<0.01). The levels of IL10 and TGFβ1 were increased in pAdeasyTM Galectin9 group compared with control group and adenovirus group(P<0.01).Conclusion pAdeasyTM Galectin9 can promote the proliferation and differentiation of CD4+CD25+ Treg cells in vivo, and can greatly improve the survival time of cardiac allograft after heart transplantation in mice.