NFATc1 has been characterized as a master regulator of RANKL-induced osteoclast differentiation. Herein, we demonstrate a novel role for NFATc1 as a positive regulator of RANKL-mediated osteoclast fusion as well as other fusion-inducing factors such as TNF-. Exogenous overexpression of a constitutively active form of NFATc1 in bone marrow-derived monocyte/macrophage cells (BMMs) induces formation of multinucleated osteoclasts as well as the expression of fusion-mediating molecules such as the d2 isoform of vacuolar ATPase V_o domain (Atp6v0d2) and the dendritic cell-specific transmembrane protein (DC-STAMP). Moreover, inactivation of NFATc1 by cyclosporin A treatment attenuates expression of Atp6v0d2 and DC-STAMP and subsequent fusion process of osteoclasts. We show that NFATc1 binds to the promoter regions of Atp6v0d2 and DC-STAMP in osteoclasts and directly induces their expression. Furthermore, overexpression of Atp6v0d2 and DC-STAMP rescues cell-cell fusion of pre-osteoclasts in spite of reduced NFATc1 activity. Our data indicate for the first time that the NFATc1/Atp6v0d2 and DC-STAMP signaling axis plays a key role in the osteoclast multinucleation process, which is essential for efficient bone resorption.