Maternal spiral artery remodeling after embryo implantation is a crucial process for successful pregnancy and rely on well-controlled trophoblast functions. Ephrin-B2 is found to be of great importance in various cell functions in both benign human tissue and tumors. However, its role in the regulation of trophoblast remains unknown. This study is conducted to investigate the role of ephrin-B2-induced trophoblast functions related to artery remodeling.Trophoblast cell line HTR-8/SVneo was used to investigate the effects of ephrin-B2 inhibition on cell proliferation, apoptosis, migration, invasion and tube formation. Placental-decidual co-culture (PDC) system was conducted to verify ephrin-B2-induced trophoblast functions ex vivo. Factors involving in artery remodeling process, such as matrix metalloproteinases (MMPs), placental growth factors (PlGF) and vascular endothelial growth factor (VEGF) were tested at transcriptional level.Inhibition of ephrin-B2 suppressed cell proliferation and induced cell apoptosis in HTR-8/SVneo cells. Down-regulation of ephrin-B2 impaired migration/invasion capabilities of HTR-8/SVneo cells and significantly decreased gene expression of MMPs. Also, a worse tube formation and a decrease in gene expression of PlGF was observed after down-regulation of ephrin-B2. However gene expression of VEGF-A did not show significantly statistical difference. These effects were further confirmed by PDC system showing an inadequate trophoblast invasion and spiral artery remodeling.Ephrin-B2 might be act as a positive regulator in maternal artery remodeling via both trophoblast invasion and endovascular formation.