CD4( )CD25( )Foxp3( ) regulatory T cells (CD25( ) Treg cells) direct the maintenance of immunological self-tolerance by active suppression of autoaggressive T-cell populations. However, the molecules mediating the anergic state and regulatory function of CD25( ) Treg cells are still elusive. Using differential proteomics, we identified galectin-10, a member of the lectin family, as constitutively expressed in human CD25( ) Treg cells, while they are nearly absent in resting and activated CD4( ) T cells. These data were confirmed on the mRNA and protein levels. Single-cell staining and flow cytometry showed a strictly intracellular expression of galectin-10 in CD25( ) Treg cells. Specific inhibition of galectin-10 restored the proliferative capacity of CD25( ) Treg cells and abrogated their suppressive function. Notably, first identified here as expressed in human T lymphocytes, galectin-10 is essential for the functional properties of CD25( ) Treg cells.