Tregs are involved in immune disorder during sepsis; they can lead to a Th2 immune reaction. Their inhibitory effects can help alleviate inflammatory injury, but may also cause secondary immune inhibition. Thymosin-alpha1 is a polypeptide with powerful immunomodulatory activities. Current reports have shown that Thymosin-alpha1 conferred beneficial effects to septic patients. To explore the relationship between Thymosin-alpha1 and Tregs, in this study, we investigated the changing trend in CD4(+)CD25(+)Foxp3(+) T lymphocytes in a CLP septic mouse model. We also investigated the variation of apoptotic rate of CD4(+)CD25(+) T lymphocytes, cytokine variation, and change of model survival rate when Thymosin-alpha1 intervening or not. We observed that the 72-h survival rate was improved, the percentage of CD4(+)CD25(+)Foxp3(+) T lymphocytes decreased and the apoptosis rate of CD4(+)CD25(+) T lymphocytes increased after intervention of Thymosin-alpha1. At same time the expression of pro-inflammation cytokines IL-2, TNF-alpha and anti-inflammatory cytokines IL-10 and TGF-beta were regulated. In conclusion, Thymosin-alpha1 can effectively control the inflammatory response intensity and improve the 72-h survival rate of septic mice. Regulating Tregs may be another important role of Thymosin-alpha1 conditioning the immune reaction in sepsis.