DCs are specialized APCs capable of inducing T cell activation as well as promoting tolerance. Although Gal, a family of β-galactoside-binding proteins, were found to affect immunity, little is known about the contribution of DC-expressed Gal on T cell activation. Here, we show that human imDCs and mDCs constitutively express Gal-1, Gal-3, Gal-8, and Gal-9 at mRNA and protein levels. Two of the most abundant Gal-Gal-1 and Gal-3-were highly expressed and detected on the cell surface of DCs. In contrast to Gal-8, knockdown of Gal-1 or Gal-3 in DCs enhanced allogeneic T cell responses. This was observed with imDCs and mDCs, but the effects were more pronounced with imDCs. Furthermore, allogeneic CD4(+) T cells incubated with Gal-1 or Gal-3 knockdown DCs produced more IFN-γ and less IL-10 than did control cells. The percentage of apoptotic T cells was significantly higher in cultures with control DCs than that with Gal-1 or Gal-3 knockdown DCs. Collectively, the data indicate that DC-expressed Gal-1 and Gal-3 are regulatory molecules that favor the inhibition of T cell activation. Furthermore, the data provide a new mechanism for the poor capacity of imDCs to stimulate T cells.