Abstract To examine the possible enrolment of Na+/K+-ATPase during osteoclast differentiation, Na+/K+-ATPase inhibitors, including ouabain and vanadate, were used in this study. These inhibitors significantly inhibited cell–cell fusion of RAW264.7 cells and bone marrow cells induced by RANKL. Interestingly, in response to RANKL-stimulation, ouabain and vanadate decreased the number of large TRAP+ osteoclasts in the culture of RAW264.7 cells, as well as bone marrow cells. In contrast, the number of small TRAP+ osteoclasts either increased in RAW264.7 cells or were otherwise less affected in bone marrow cells than large TRAP+ osteoclasts. Large TRAP+ osteoclasts are defined as having ≥10 nuclei/cell and having more potency in bone resorption than small multinuclear osteoclasts with <9 nuclei/cell. Na+/K+-ATPase α1 and β2 mRNAs were detected in sRANKL-stimulated RAW264.7 cells. Moreover, real-time quantitative PCR showed that ouabain and vanadate suppressed the RANKL-dependent induction of the osteoclast fusion-promotion molecule DC-STAMP at the mRNA level. Finally, and importantly, RNAi-mediated suppression of Na+/K+-ATPase α1 resulted in a diminished number of large TRAP+ osteoclasts in the sRANKL-stimulated RAW264.7 cells, along with the decreased level of DC-STAMP mRNA expression. These findings strongly suggest that blockage of the Na+/K+-ATPase α1 subunit by ouabain or vanadate caused the inhibition of RANKL-induced cell–cell fusion, resulting in the generation of large osteoclasts through suppression of DC-STAMP expression. Thus, in addition to its known function of sodium and potassium ion exchange during bone resorption by mature osteoclasts, this study has revealed a novel molecular role of the Na+/K+-ATPase α1 subunit in osteoclastogenesis.