Objective：To explore an induced and culture method for CD4+CD25+Treg cells in vitro，study the effect of Zerumbone about the differentiation and secretion functions of Treg cells and explore the mechanisms included. Methods：CD4+CD62L+T cells were isolated from BALB/c mice spleen and purified with magnetic bead methods. CD4+CD62L+T cells were co-cultured with transforming growth factor（TGF）-beta（5 ng/mL），interleukin（IL）-2（30 μg/mL） for CD4+CD25+Treg polarization．The cultured CD4+CD25+Treg cells were divided into five groups：the normal group；the induced group，which were cultured with the above protocol；Zerumbone（1 μmol/L）group；Zerumbone（10 μmol/L）group； Zerumbone（30 μmol/L）group. Flow cytometry was used to detect the proportion of CD4+CD25+Treg cells. The ELISA method was detected the levels of IL-10. Reverse transcriptase polymerase chain reaction（RT-PCR）was detected the level of IL-10 mRNA and Foxp3 mRNA. Results：The proportion of CD4+CD25+Treg cells cultured with the protocol were significantly higher compared with the normal group（P < 0.05）. The CD4+CD25+Treg cells proportion in Zerumbone（1 μmol/L），Zerumbone（10 μmol/L），Zerumbone（30 μmol/L）groups were significantly increased compared with group model，there is dose dependent（P < 0.05）. The protein level of IL-10 was increased by Zerumbone and that was also dose-dependent. Zerumbone increased the expression of IL-10 mRNA and Foxp3 mRNA（P < 0.05）. Conclusion：Zerumbone can increase the differentiation of splenic CD4+CD62L Zerumbone into CD4+CD25+Treg cells and induce the expressions of IL-10 protein in vitro. The results may be thought the activation of Foxp3 in CD4+CD25+Treg cells.