ABSTRACT Angiogenesis is a crucial defense response to hypoxia that regulates the process of raising the promise of long‐term neurologic recovery during the management of stroke. A high expression of antiangiogenic factors leads to the loss of neovascularization capacity in pathologic conditions. We have previously documented an impairment of the cerebral vessel perfusion and neovascularization in the cortex neighboring the stroke‐induced lesion, which was accompanied by an activation of semaphorin 3E (Sema3E)/PlexinD1 after ischemic stroke. In this study, we employed micro‐optical sectioning tomography to fully investigate the details of the vascular pattern, including the capillaries. We found that after transient middle cerebral artery occlusion, inhibiting PlexinD1 signaling led to an organized recovery of the vascular network in the ischemic area. We then further explored the possible mechanisms. In vivo , Sema3E substantially decreased dynamic delta‐like 4 (DLL4) expression. In cultured brain microvascular endothelial cells, Sema3E down‐regulated DLL4 expression via inhibiting Ras‐related C3 botulinum toxin substrate 1–induced JNK phosphorylation. At the microcosmic level, Sema3E/PlexinD1 signaling promoted F‐actin disassembly and focal adhesion reduction by activating the small guanosine triphosphatase Ras homolog family member J by releasing RhoGEF Tuba from direct binding to PlexinD1, thus mediating endothelial cell motility and filopodia retraction. Our study reveals that Sema3E/PlexinD1 signaling, which suppressed endothelial DLL4 expression, cell motility, and filopodia formation, is expected to be a novel druggable target for angiogenesis during poststroke progression.— Zhou, Y.‐F., Chen, A.‐Q., Wu, J.‐H., Mao, L., Xia, Y.‐P., Jin, H.‐J., He, Q.‐W., Miao, Q. R., Yue, Z.‐Y., Liu, X.‐L., Huang, M., Li, Y.‐N., Hu, B. Sema3E/PlexinD1 signaling inhibits postischemic angiogenesis by regulating endothelial DLL4 and filopodia formation in a rat model of ischemic stroke. FASEB J. 33, 4947–4961 (2019). www.fasebj.org