The ephB4-ephrinB2 system plays an important role in the interaction of tumor cells with endothelial cells (ECs). To assess the role of ephB4 in the in vivo growth of head and neck squamous cell carcinoma (HNSCC), we used ephrinB2-Fc, a fusion protein consisting of the extracellular domain of ephrin-B2 and the Fc portion of human IgG1, as the soluble ligand for ephB4. EphrinB2-Fc injection into HNSCC xenografted mice significantly suppressed xenograft growth, accompanied by a decrease in vessel cross-sectional area, but there was no change in vessel number. EphrinB2-Fc injection also induced the formation of mature blood vessels rich in alpha-smooth muscle actin positive pericytes in the xenograft tissue. In vitro assays revealed that ephrinB2-Fc inhibited the proliferation of human umbilical vein ECs (HUVECs) but not tumor cells. Furthermore, real-time quantitative RT-PCR showed that ephrinB2-Fc down-regulated matrix metalloproteinase-2 mRNA expression in HUVECs and vascular endothelial growth factor-A in tumor cells. These data suggest that treatment with ephrinB2-Fc, the soluble ligand of ephB4, inhibited the growth of HNSCC through vessel maturation/stabilization, preventing leakiness and endothelial sprout formation.