A critical control point in the immune response to tumors or to pathogens is the egress of lymphocytes from blood into damaged or infected tissue. While several specific endothelial cell proteins promote lymphocyte adhesion to and migration across endothelium, little is known about endothelial cell surface proteins that negatively regulate transendothelial migration of lymphocytes. Galectin-1 is a mammalian lectin expressed by a variety of cell types, including endothelial cells, that has pleiotropic anti-inflammatory effects. Galectin-1 is known to alter T-cell cytokine production and to trigger T-cell death. We now demonstrate that galectin-1 inhibits T-cell migration across endothelial cells, identifying a novel anti-inflammatory effect of galectin-1. We observed reduced T-cell migration across endothelial cells induced to increase galectin-1 expression by exposure to prostate cancer cell conditioned medium, compared to T-cell migration across control-treated endothelial cells, and the inhibitory effect of galectin-1 on T-cell migration was reversed by specific antiserum. Decreased T-cell migration was not due to decreased adhesion to galectin-1 expressing endothelial cells, nor to death of T cells, as T cells lacking core 2 O-glycans and thus resistant to galectin-1 death displayed reduced migration across endothelial cells. Galectin-1 on the surface of extracellular matrix also reduced the ability of T cells to migrate through the matrix. T cells bound to galectin-1-coated matrix demonstrated enhanced clustering of CD43, including at the T-cell:matrix interface, compared to CD43 on T cells bound to matrix in the absence of galectin-1. As translocation of CD43 to the trailing edge is essential for polarized T-cell migration, these data indicate that galectin-1-mediated clustering of CD43 contributes to the inhibitory effect on T-cell migration. Inhibition of T-cell migration is a novel anti-inflammatory activity of galectin-1.