The trigger for bone remodeling is bone resorption by osteoclasts. Osteoclast differentiation only occurs on the old bone, which needs to be repaired under physiological conditions. However, uncontrolled bone resorption is often observed in pro‐inflammatory bone diseases, such as rheumatoid arthritis. Mature osteoclasts are multinuclear cells that differentiate from monocyte/macrophage lineage cells by cell fusion. Although Osteoclast precursors should migrate across osteoblast layer to reach bone matrix before maturation, the underlying mechanisms have not yet been elucidated in detail. We herein found that osteoclast precursors utilize two routes to migrate across osteoblast layer by confocal‐ and electro‐microscopic observations. The osteoclast supporting activity of osteoblasts inversely correlated with osteoblast density and was positively related to the number of osteoclast precursors under the osteoblast layer. Osteoclast differentiation was induced by IL‐1ß, but not by PGE2 in high‐density osteoblasts. Osteoblasts and osteoclast precursors expressed CX3CL1 and CX3CR1, respectively, and the expression of CX3CL1 increased in response to interleukin‐1ß. An anti‐CX3CL1‐neutralizing antibody inhibited the migration of osteoclast precursors and osteoclast differentiation. These results strongly suggest the involvement of CX3CL1 in the migration of osteoclast precursors and osteoclastogenesis, and will contribute to the development of new therapies for bone diseases. J. Cell. Physiol. 232: 1739–1745, 2017. © 2016 Wiley Periodicals, Inc. Transcellular and paracellular migration of osteoclast precursors across osteoblast layer were observed. CX3CL1 was involved in the migration of osteoclast precursors and osteoclastogenesis.