The TCIRG1 gene encodes the a3 isoform of vacuolar H+-ATPase (V-ATPase), which forms a proton transport channel in osteoclasts. Defects in this gene lead to functional impairment of osteoclasts and increased bone mass; however, the molecular mechanisms of TCIRG1 loss have not been fully elucidated. In the current study, we transfected mouse bone marrow-derived monocytes with control or Tcirg1-knockdown lentiviruses to further investigate the mechanisms of TCIRG1. Our results demonstrate that knockdown of Tcirg1 inhibits large-osteoclast (>100 μm) generation by decreasing the expression of nuclear factor of activated T-cells 1 (NFATc1) and inositol-1,4,5-trisphosphate receptor 2 (IP3R2). The decreased IP3R2 reduces intracellular calcium levels, which limits the nuclear translocation of NFATc1 in RANKL-induced mouse bone marrow-derived monocytes. These findings provide a mechanism to explain the effects of TCIRG1 impairment, with potential implications for the development of therapies for osteopetrosis.