Tregs play a crucial role in modulating the inflammatory response and participated in the sepsis-related immune dysfunctions. However, little is known about the regulatory mechanisms by which Tregs are kept in check during immune responses. Here, we verified the simultaneous expression of IL-3 and its receptor (IL-3R) on Tregs. Then, by modulation of IL-3 expression via lentiviral transduction mediated siRNA, we demonstrated that IL-3 negatively regulated Tregs activity via an autocrine mechanism. Furthermore, we found that anti-IL-3 antibody treatment significantly diminished inflammatory cytokines and organ injury, and improved the survival in septic mice, which was associated with enhanced Tregs percentage and functions. Collectively, these results suggest that IL-3 negatively regulates the activity of Tregs by a previously unrevealed autocrine manner, and plays an important role in the excessive inflammatory response in sepsis, which might be utilized as a therapeutic strategy for the treatment of septic complications.© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: email@example.com.