Osteoclasts are absorptive cells that play a critical role in homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role of epigenetic regulation in osteoclastogenesis. In this study, we investigated the role of DOT1L, which regulates gene expression epigenetically by histone H3K79 methylation (H3K79me), during osteoclast formation. Using RANKL-induced RAW264.7 macrophage cells as an osteoclast differentiation model, we found that DOT1L and H3K79me2 levels were upregulated during osteoclast differentiation. Small molecule inhibitor- (EPZ5676 or EPZ004777) or short hairpin RNA-mediated reduction in DOT1L expression promoted osteoclast differentiation and resorption. In addition, DOT1L inhibition increased osteoclast surface area and accelerated bone-mass reduction in a mouse ovariectomy (OVX) model of osteoporosis without alter osteoblast differentiation. DOT1L inhibition increase reactive oxygen species (ROS) generation and autophagy activity, and cell migration in pre-osteoclasts. Moreover, it strengthened expression of osteoclast fusion and resorption-related protein CD9 and MMP9 in osteoclasts derived from RAW264.7. Our findings support a new mechanism of DOT1L-regulated, H3K79me2-mediated, epigenetic regulation of osteoclast differentiation, implicating DOT1L as a new therapeutic target for osteoclast dysregulation-induced disease.