The naturally occurring population of dedicated regulatory T cells that coexpress CD4 and CD25 is known to play a key role in the maintenance of peripheral T-cell tolerance; however, their mechanism of action has remained obscure. Here we report that a member of the family of β-galactoside–binding proteins, galectin-1, is overexpressed in regulatory T cells, and that expression is increased after activation. Most importantly, blockade of galectin-1 binding significantly reduced the inhibitory effects of human and mouse CD4⁺CD25⁺ T cells. Reduced regulatory activity was observed in CD4⁺CD25⁺ T cells obtained from galectin-1–homozygous null mutant mice. These results suggest that galectin-1 is a key effector of the regulation mediated by these cells.