Bisphosphonates are therapeutic agents in the treatment of post-menopausal osteoporosis. Although they have been associated with delayed healing in injured tissues, inappropriate femoral fractures, and osteonecrosis of the jaw (ONJ), the pathophysiological mechanisms involved are not clear. Our hypothesis is that alendronate, a member of the N-containing bisphosphonates, indirectly inhibits osteoblast function through the coupling of osteoclasts to osteoblasts by ephrinB-EphB interaction. We found that alendronate increased gene and protein expression of ephrinB1 and EphB1, as well as B3, in femurs of adult mice injected with alendronate (10 µg/100 g/wk) for 8 weeks. Alendronate suppressed the expression of bone sialoprotein (BSP) and osteonectin in both femurs and bone marrow osteoblastic cells of mice. After elimination of pre-osteoclasts from bone marrow cells, alendronate did not affect osteoblast differentiation, indicating the need for pre-osteoclasts for alendronate's effects. Alendronate stimulated EphB1 and EphB3 protein expression in osteoblasts, whereas it enhanced ephrinB1 protein in pre-osteoclasts. In addition, a reverse signal by ephrinB1 inhibited osteoblast differentiation and suppressed BSP gene expression. Thus, alendronate, through its direct effects on the pre-osteoclast, appears to regulate expression of ephrinB1, which regulates and acts through the EphB1, B3 receptors on the osteoblast to suppress osteoblast differentiation.