Niclosamide is a potent inhibitor of osteoclastogenesis and bone remodeling. DK-520 is an acyl derivative of Niclosamide and significantly increased both the plasma concentration and the duration of exposure of Niclosamide when dosed orally. However, at present the effect of DK-520 on osteoclastogenesis has not been reported. Here, we investigated whether DK-520 can regulate receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis of bone marrow macrophages (BMMs) in vitro. Following induction of BMMs with RANKL for three days, we detected differentiated osteoclasts with typical morphology and high levels of tartrate-resistant acid phosphatase (TRAP), RANKL, and cathepsin K (CTSK) expression. Treatment with either Niclosamide or DK-520 did not affect the viability of osteoclast precursors (OCPs), but significantly inhibited RANKL-induced transdifferentiation of macrophages into OCPs, particularly in the early stage of osteoclastogenesis. Both Niclosamide and DK-520 significantly decreased the relative levels of transcription factor PU.1 mRNA transcripts and dendritic cell-specific transmembrane protein (DC-STAMP), but not v-ATPasev0d2 protein expression in OCPs. In addition, the inhibitory effect of DK-520 on osteoclastogenesis is realized through impairment of the NF-kB (nuclear factor-κB) and MAPK (mitogen-activated protein kinase) signaling pathways. These results demonstrate that DK-520, like Niclosamide, effectively inhibits the early stage of osteoclastogenesis. The findings presented here, together with its increased oral plasma concentrations and bioavailability, suggest that DK-520 may be a promising drug candidate for treatment of osteoclast-related diseases.