CD4+CD25+ regulatory T cells (Tregs) play an essential role in the suppression of the immune response and prevention of autoimmune reactions. The activation of TLR4, which provides a critical link between the innate and adaptive immune systems, has been implicated in regulating the function of Tregs. Ulinastatin (UTI) is a broad-spectrum protease inhibitor that has been shown to modulate innate immunity and pro-inflammatory signaling in sepsis. In addition, there are reports that UTI may modulate the functional activity of Tregs to influence the inflammatory response in infectious disease. In the present study, we investigated the effect of UTI on the activity of Tregs, which was assessed by measuring the survival and inflammatory responses of mice with cecal ligation and puncture (CLP)-induced sepsis. In addition, we further explored the cellular and molecular mechanisms involved in these effects. The results showed that UTI could enhance survival and attenuate inflammatory responses during CLP-induced sepsis. Moreover, sepsis-induced increases in the quantity and activity of Tregs were attenuated under UTI treatment, but not in TLR4-/- mice. We also found that the functional changes in Tregs could be attributed to the TLR4/NF-κB signaling pathway. Collectively, our results indicated that UTI could ameliorate inflammatory damage by modulating the quantity and function of Tregs via the TLR4/NF-κB signaling pathway. Our study provides theoretical and experimental evidence for the administration of UTI in the treatment of sepsis and other acute critical illnesses.