Normal bone mass is maintained by balanced bone formation and resorption. Myosin X (Myo10), an unconventional "myosin tail homology 4-band 4.1, ezrin, radixin, moesin" (MyTH4-FERM) domain containing myosin, is implicated in regulating osteoclast (OC) adhesion, podosome positioning, and differentiation in vitro. However, evidence is lacking for Myo10 in vivo function. Here we show that mice with Myo10 loss of function, Myo10(m/m), exhibit osteoporotic deficits, which are likely due to the increased OC genesis and bone resorption because bone formation is unchanged. Similar deficits are detected in OC-selective Myo10 conditional knockout (cko) mice, indicating a cell autonomous function of Myo10. Further mechanistic studies suggest that Unc-5 Netrin receptor B (Unc5b) protein levels, in particular its cell surface level, are higher in the mutant OCs, but lower in RAW264.7 cells or HEK293 cells expressing Myo10. Suppressing Unc5b expression in bone marrow macrophages (BMMs) from Myo10(m/m) mice by infection with lentivirus of Unc5b shRNA markedly impaired RANKL-induced OC genesis. Netrin-1, a ligand of Unc5b, increased RANKL-induced OC formation in BMMs from both wild-type and Myo10(m/m) mice. Taken together, these results suggest that Myo10 plays a negative role in OC formation, likely by inhibiting Unc5b cell-surface targeting, and suppressing Netrin-1 promoted OC genesis. (c) 2019 American Society for Bone and Mineral Research.