Abstract: In spite of their pivotal role in orchestrating immunity, dendritic cells (DCs) may be licensed by immunosuppressive signals to become tolerogenic. Here we show that ligation of cell surface glyco-receptors by Galectin-1, an endogenous glycan-binding protein, can drive the differentiation of regulatory DCs with tolerogenic potential in vivo. Galectin-1-differentiated DCs acquired a tolerogenic phenotype characterized by IL-27-dependent, STAT3-mediated and CD45RB+IL-10+ regulatory signatures. Adoptive transfer of galectin-1-conditioned DCs induced T-cell tolerance in inflammatory and neoplastic settings and dampened TH1- and TH-17-mediated autoimmune neuroinflammation. Consistent with a negative regulatory function of endogenous galectin-1, DCs from galectin-1-deficient (Lgals1?/?) mice had greater immunogenic capacity compared with their wild-type counterparts.