Regulatory T cells are critical in the modulation of immune responses. Accordingly, the manipulation and implementation of in vitro induced Treg cells to control immune function has a significant potential in clinical medicine. However, the use of iTregs as therapeutics is complicated by their poor stability and variable suppressive activity. To gain a better understanding of the molecular characteristics of human iTreg cells, we conducted a data-independent analysis-proteomics, (detecting over 8000 proteins and providing quantitative comparison of their relative amounts in iTreg and activated Th0 cells). Consistent with the known molecular characteristics of Tregs, a full complement of Treg signature proteins, including FOXP3, EOS, IL2RA, CTLA4, PDCD1, IKZF3, LAG3, RUNX1 and HIC1were identified and validated using targeted proteomics and/or TaqMan assay. In addition, elevated levels of LPXN were observed during Treg cell differentiation. As a follow to the latter finding, we observed that LPXN deficient cells showed impaired expression of Treg protein markers, FOXP3, EOS, and IKZF3 and reduced ability to suppress effector cells. Collectively, this study characterized the proteomic signature of human iTregs and identified a novel role of LPXN in the development and suppressive activity of Tregs.