The identification of new signaling pathways critical for cardiac morphogenesis will contribute to our understanding of congenital heart disease (CHD), which remains a leading cause of mortality in newborn children worldwide. Signals mediated by semaphorin ligands and plexin receptors contribute to the intricate patterning of axons in the central nervous system. Here, we describe a related signaling pathway involving secreted class 3 semaphorins, neuropilins, and a plexin receptor, PlexinD1, expressed by endothelial cells. Interruption of this pathway in mice results in CHD and vascular patterning defects. The type of CHD caused by inactivation of PlexinD1 has previously been attributed to abnormalities of neural crest. Here, we show that this form of CHD can be caused by cell-autonomous endothelial defects. Thus, molecular programs that mediate axon guidance in the central nervous system also function in endothelial cells to orchestrate critical aspects of cardiac morphogenesis.