Regulatory T cells (Tregs) play a central role in maintaining immune homeostasis, and the implementation of in vitro induced Treg cells (iTreg) to control immune function has significant potential in clinical medicine. The clinical application of iTregs has been limited by their poor stability. To better define the molecular characteristics of human iTregs, we performed a data-independent acquisition proteomics, detecting over 8000 proteins and providing a quantitative comparison of their relative levels in iTregs and activated Th0 cells. Consistent with the known molecular characteristics of Tregs, several Treg signature proteins, including FOXP3, Eos, IL2RA, CTLA4, PD-1, Aiolos, LAG3, RUNX1 and HIC1, were identified and validated using targeted proteomics and/or qRT-PCR. Notably, leupaxin (LPXN) level was upregulated during Treg cell differentiation. Functional studies demonstrated that LPXN-deficient cells showed impaired expression of Treg protein markers FOXP3, Eos and IKZF3 and impaired suppression of effector T cells. In addition, we identified a distinct CD160⁺ iTregs subpopulation characterized by a distinct proteomic signature as compared to CD160⁻ iTregs. Together, these findings provide a high-resolution proteomic landscape of human iTregs and identified a novel role of LPXN in the development and suppressive activity of iTregs.