The recruitment of osteoclast precursors toward osteoblasts and subsequent cell-cell interactions are critical for osteoclast differentiation. Chemokines are known to regulate cell migration and adhesion. CX3CL1 (also called fractalkine) is a unique membrane-bound chemokine that has dual functions for cells expressing its receptor CX3CR1: a potent chemotactic factor in its soluble form and a type of efficient cell adhesion molecule in its membrane-bound form. In this paper, we demonstrate a novel role of CX3CL1 in osteoblast-induced osteoclast differentiation. We found that osteoclast precursors selectively expressed CX3CR1, whereas CX3CL1 is expressed by osteoblasts. We confirmed that soluble CX3CL1 induced migration of bone marrow cells containing osteoclast precursors, whereas immobilized CX3CL1 mediated firm adhesion of osteoclast precursors. Furthermore, a blocking mAb against CX3CL1 efficiently inhibited osteoclast differentiation in mouse bone marrow cells cocultured with osteoblasts. Anti-CX3CL1 also significantly suppressed bone resorption in neonatal mice by reducing the number of bone-resorbing mature osteoclasts. Collectively, CX3CL1 expressed by osteoblasts plays an important role in osteoclast differentiation, possibly through its dual functions as a chemotactic factor and adhesion molecule for osteoclast precursors expressing CX3CR1. The CX3CL1-CX3CR1 axis may be a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis, osteoporosis, and cancer bone metastasis.