Osteocytes are terminally differentiated osteoblasts embedded in the bone matrix. Evidence indicates that cells in the mesenchymal lineage possess plasticity. However, whether or not osteocytes have the capacity to dedifferentiate back into osteoblasts is unclear. This study aimed to clarify the dedifferentiation potential of osteocytes. Mouse calvarial osteoblasts were isolated and maintained in normal two-dimensional (2D) or collagen gel three-dimensional (3D) cultures. In 2D cultures, osteoblasts exhibited a typical fibroblast-like shape with high Alpl and minimal Sost, Fgf23, and Dmp1 expression and osteoblasts formed mineralised nodules. When these osteoblasts were transferred into 3D cultures, they showed a stellate shape with diminished cytoplasm and numerous long processes and expression of Alpl decreased while Sost, Fgf23, and Dmp1 were significantly increased. These cells were in cell cycle arrest and showed suppressed mineralisation, indicating that they were osteocytes. When these osteocytes were recovered from 3D cultures and cultured two-dimensionally again, they regained adequate cytoplasm and lost the long processes, resulting in a fibroblast-like shape. These cells showed high Alpl and low Sost, Fgf23, and Dmp1 expression with a high mineralisation capability, indicating that they were osteoblasts. This report shows that osteocytes possess the capacity to dedifferentiate back into mature osteoblasts without gene manipulation.