Clarification of the mechanisms underlying osteoclast differentiation enables us to understand the physiology of bone metabolism as well as the pathophysiology of bone diseases such as osteoporosis. Recently, it has been reported that epigenetics can determine cell fate and regulate cell type–specific gene expression. However, little is known about epigenetics during osteoclastogenesis. To reveal a part of epigenetics, especially focused on chromatin dynamics, during early osteoclastogenesis and to identify novel transcription factors involved in osteoclastogenesis, we performed a genome-wide analysis of open chromatin during receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis using DNase I hypersensitive sites sequencing (DNase-seq). DNase-seq was performed using the extracted nuclei from RAW264 cells treated with or without RANKL for 24 hours, followed by several bioinformatic analyses. DNase I hypersensitive sites (DHSs) were dynamically changed during RANKL-induced osteoclastogenesis and they accumulated in promoter regions. The distributions of DHSs among cis-regulatory DNA regions were identical regardless of RANKL stimulation. Motif discovery analysis successfully identified well-known osteoclastogenic transcription factors including Jun, CREB1, FOS, ATF2, and ATF4, but also novel transcription factors for osteoclastogenesis such as Zscan10, Atf1, Nrf1, and Srebf2. siRNA knockdown of these identified novel transcription factors impaired osteoclastogenesis. Taken together, DNase-seq is a useful tool for comprehension of epigenetics, especially chromatin dynamics during osteoclastogenesis and for identification of novel transcription factors involved in osteoclastogenesis. This study may reveal underlying mechanisms that determine cell type–specific differentiation of bone cells and may lead to investigation of novel therapeutic targets for osteoporosis. © 2014 American Society for Bone and Mineral Research.