Cholesterol is one of the major components of biological membranes and has an important function in osteoclast formation and survival. It has been reported that high-density lipoprotein (HDL) promotes cholesterol efflux from osteoclasts and induces their apoptosis, but the underlying mechanisms are unclear. In this study, we investigated how HDL promotes osteoclast cholesterol efflux and explored its effect on osteoclast formation and survival. Our results showed that the maximum diameter and fusion index of osteoclasts were decreased, while the ratios of osteoclasts with pyknotic nuclei were increased when cells were treated with HDL (600 ng/ml), as revealed by tartrate-resistant acid phosphatase-positive staining and microscopy assay. HDL enhanced cellular cholesterol efflux from osteoclasts in both concentration-and time-dependent manners. The ability of HDL3 to stimulate cholesterol efflux was stronger than preβ-HDL, HDL2, and ApoAI. Knockdown of ABCG1 expression reduced HDL-mediated cholesterol efflux and restored the HDL-induced reduction in osteoclast formation. Finally, HDL3 promoted sphingomyelin efflux from osteoclasts and reduced the expression of caveolin-1. Together, the findings demonstrate that HDL3 upregulates ABCG1 expression and promotes cholesterol efflux from osteoclast, impairs cholesterol homeostasis in osteoclasts, and consequently enhances osteoclast apoptosis.