Macrophage colony-stimulating factor (M-CSF) and interleukin-34 (IL-34) are ligands for the colony-stimulating factor-1 receptor (CSF-1r) expressed on the surface of monocyte/macrophage lineage cells. The importance of coordinated signaling between M-CSF/receptor activator of the nuclear factor kappa-Β ligand (RANKL) in physiological and pathological bone remodeling and alveolar bone loss in response to oral bacterial colonization is well established. However, our knowledge about the IL-34/RANKL signaling in periodontal bone loss remains limited. Recently published cohort studies have demonstrated that the expression patterns of IL-34 are dramatically elevated in gingival crevicular fluid collected from patients with periodontitis. Therefore, the present study aims to evaluate the effects of IL-34 on osteoclastogenesis in vitro and in experimental ligature-mediated model of periodontitis using male mice. Our initial in vitro study demonstrated increased RANKL-induced osteoclastogenesis of IL-34-primed osteoclast precursors (OCPs) compared to M-CSF-primed OCPs. Using an experimental model of ligature-mediated periodontitis, we further demonstrated elevated expression of IL-34 in periodontal lesions. In contrast, M-CSF levels were dramatically reduced in these periodontal lesions. Furthermore, local injections of mouse recombinant IL-34 protein significantly elevated cathepsin K activity, increased the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and promoted alveolar bone loss in periodontitis lesions. In contrast, anti-IL-34 neutralizing monoclonal antibody significantly reduced the level of alveolar bone loss and the number of TRAP-positive osteoclasts in periodontitis lesions. No beneficial effects of locally injected anti-M-CSF neutralizing antibody were observed in periodontal lesions. This study illustrates the role of IL-34 in promoting alveolar bone loss in periodontal lesions and proposes the potential of anti-IL34 monoclonal antibody (mAb)-based therapeutic regimens to suppress alveolar bone loss in periodontitis lesions.