Osteoclasts are the principal bone resorption cells crucial for homeostatic bone remodeling and pathological bone destruction. Increasing data demonstrate a vital role of histone methylation in osteoclastogenesis. As an integral core subunit of H3K4 methyltransferases, Dpy30 is notal as a key chromatin regulator for cell growth and differentiation and stem cell fate determination, particularly in the hematopoietic system. However, its role in osteoclastogenesis is currently unknown. Herein, we generated Dpy30F/F; LysM-Cre+/+ mice, which deletes Dpy30 in myeloid cells, to characterize its involvement in osteoclast differentiation and function. Dpy30F/F; LysM-Cre+/+ mice showed increased bone mass, evident by impaired osteoclastogenesis and defective osteoclast activity, but no alteration of osteoblast numbers and bone formation. Additionally, our ex vivo analysis showed that the loss of Dpy30 significantly impedes osteoclast differentiation and suppresses osteoclast-related gene expression. Moreover, Dpy30 deficiency significantly decreased the enrichment of H3K4me3 on the promoter region of NFATc1. Thus, we revealed a novel role for Dpy30 in osteoclastogenesis through epigenetic mechanisms, and that it could potentially be a therapeutic target for bone destruction diseases.