RANKL (receptor activator of NF-kappaB ligand) induces osteoclastogenesis by activating multiple signaling pathways in osteoclast precursor cells, chief among which is induction of long lasting oscillations in the intracellular concentration of Ca(2+) ([Ca(2+)](i)). The [Ca(2+)](i) oscillations activate calcineurin, which activates the transcription factor NFATc1. The pathway by which RANKL induces [Ca(2+)](i) oscillations and osteoclastogenesis is poorly understood. Here we report the discovery of a novel pathway induced by RANKL to cause a long lasting increase in reactive oxygen species (ROS) and [Ca(2+)](i) oscillations that is essential for differentiation of bone marrow-derived monocytes into osteoclasts. The pathway includes RANKL-mediated stimulation of Rac1 to generate ROS, which stimulate phospholipase Cgamma1 to evoke [Ca(2+)](i) oscillations by stimulating Ca(2+) release from the inositol 1,4,5-trisphosphate pool and STIM1-regulated Ca(2+) influx. Induction and activation of the pathway is observed only after 24-h stimulation with RANKL and lasts for at least 3 days. The physiological role of the pathway is demonstrated in mice with deletion of the Peroxiredoxin II gene and results in a mark increase is ROS and, consequently, a decrease in bone density. Moreover, bone marrow-derived monocytes in PrxII(-/-) primary culture show increased ROS and spontaneous [Ca(2+)](i) oscillations. These findings identify the primary RANKL-stimulated pathway to trigger the late stages of osteoclastogenesis and regulate bone resorption.