Objective To investigate the effect of diosgenin on CD4⁺CD25^-T cells of DBA1/J mice. Methods CD4⁺CD25^-T cells of DBA1/J mice were separated with immunomagnetic beads. Then, CD4⁺CD25^-T cell purity was detected by flow cytometry and culture them in vitro. After treated with diosgenin, the concentration of IL-10 was detected by ELISA, the level of transcription factor Foxp3 mRNA was detected by real-time PCR, and the proportion of Treg cells was detected by FCM. Results This study separated CD4⁺CD25^-T cells of 93.89% purity. Compared with the control group, the concentration of IL-10 in cell supernatant, the Foxp3 mRNA level and percentage of Treg cells were upregulated significantly (P<0.05) after treated with diosgenin for 48h. Conclusion Diosgenin can significantly upregulate the levels of IL-10 and Foxp3 mRNA, the proportion of Treg cells in DBA1/J mice.