EphrinB2 is expressed in osteoclasts and functions as a coupling factor between osteoblasts and osteoclasts. In this study, EphrinB2 was systemically administered to ovariectomized (OVX) mice to verify its effects on bone loss and the mechanisms. EphrinB2 administration significantly suppressed OVX-induced bone loss, and EphrinB2 increased the number of osteoblasts and decreased the number of osteoclasts on the surface of trabecular bone. EphrinB2 inhibited the osteoclast differentiation of mouse bone marrow cells, mouse bone marrow-derived macrophages, and RAW264.7 to a similar extent in vitro. Since the inhibitory effect of osteoclast differentiation was not accompanied by suppression of gene expression of osteoclast markers such as ACP5, CTSK, and DC-STAMP, it was considered that cell-cell fusion for the maturation of multinucleated osteoclasts was suppressed. On the other hand, EphrinB2 promoted osteoblast differentiation of bone marrow cells, but not osteoprogenitor cells such as calvarial cells and bone marrow-derived stromal cells. This means that EphrinB2 indirectly promotes osteoblast differentiation by acting on cells other than osteoprogenitor cells contained in the bone marrow. EphrinB2 increased the expression of BMP2 in osteoclast progenitor cells, RAW264.7, suggesting the possibility that BMP2 is involved in the action of EphrinB2 to promote osteoblast differentiation. Taken together, these results indicate that EphrinB2 can be used as a novel osteoporosis therapeutic agent having a dual effect.