Successful pregnancy depends on well-regulated extravillous trophoblast (EVT) invasion into the uterine decidua and moderate uterine spiral artery remodeling. Ephrin receptor B4 (EPHB4) is a membrane-anchored receptor tyrosine kinase that plays an important role in various cellular functions in human normal tissue and tumors. Reportedly, EPHB4 plays important roles during placentation. Still, there is no investigation of EPHB4 modulating trophoblast function. In our study, term placentas of preeclamptic pregnancies showed a significantly increased EPHB4 expression compared to those of uncomplicated pregnancies (n = 15). Exogenous up-regulation of EPHB4 in HTR-8/SVneo cells was performed to investigate the effects of EPHB4 on cell biological behavior. The results showed that EPHB4 enhancement reduced cell proliferation and promoted trophoblast apoptosis; and inhibited cell migration, invasion, and endothelial replacement. Associated factors, such as matrix metalloproteinases, vascular endothelial growth factor, placental growth factor, and soluble Fms-like tyrosine kinase 1 were examined at transcriptional level. Furthermore, cell functional results were confirmed in a placenta-decidua coculture system, showing poor vascular remodeling. Additionally, we detected possible down-stream PI3K-Akt signal pathway involved in EPHB4-mediated function of HTR-8/SVneo cells. Our study demonstrates that EPHB4 overexpression may contribute to trophoblasts dysfunction and impair maternal artery remodeling, as is associated with the pathogenesis of preeclampsia.