Originally identified as axon guidance molecules, semaphorins are now known to be widely expressed mediators that play significant roles in immune responses and organ morphogenesis. However, not much is known about the signaling pathways via which they exert their organ-specific effects. Here we demonstrate that Sema4A, previously identified as an activator of T-cell-mediated immunity, is expressed in endothelial cells, where it suppresses vascular endothelial growth factor (VEGF)-mediated endothelial cell migration and proliferation in vitro and angiogenesis in vivo. Mice lacking Sema4A exhibit enhanced angiogenesis in response to VEGF or inflammatory stimuli. In addition, binding and functional experiments revealed Plexin-D1 to be a receptor for Sema4A on endothelial cells, indicating that Sema4A exerts organ-specific activities via different receptor-mediated signaling pathways: via Plexin-D1 in the endothelial cells and via T-cell immunoglobulin and mucin domain-2 in T cells. The effects of Sema4A on endothelial cells are dependent on its ability to suppress VEGF-mediated Rac activation and integrin-dependent cell adhesion. It thus appears that Sema4A-Plexin-D1 signaling negatively regulates angiogenesis.