Objective To investigated the function and the target gene of miR-216b in osteoclast differentiation and explored its effect on osteoclast cholesterol efflux.Methods The cell model of RAW264.7 osteoclast precursor cell differentiation induced by RANKL stimulation was established. Tartrate-resistant acid phosphatase (TRAP) staining assay was conducted to evaluate osteoclasts differentiation. MiR-216b target gene, ABCG1 3′ untranslated region (3'UTR) sequence and free energy were predicted and analyzed by bioinformatics analyses and dual-luciferase reporter assays. MiR-216b mimic or inhibitor transfection was performed to verify the role of miR-216b in osteoclast differentiation. Liquid scintillation counting was used to measure [³H]-labeled cholesterol efflux from RAW264.7 macrophage-derived osteoclasts. The lipid accumulation in RAW264.7 macrophages was detected by high performance liquid chromatography (HPLC). Real-time quantitative PCR (RT-qPCR) and Western blot assays were used to assess the transcriptional and post-transcriptional levels of ABCG1 in osteoclasts.Results Our results showed that the number of osteoclasts, the average diameter of osteoclasts and the fusion index were significantly increased when cells were transfected with miR-216b mimic, as revealed by tartrate-resistant acid phosphatase-positive staining and microscopy assay. MiR-216b inhibitor showed the complete opposite outcome which brought additional evidence to our findings. Bioinformatics analysis and dual-luciferase reporter assays showed that miR-216b targets the 3′UTR of ABCG1. Moreover, miR-216b suppressed both the mRNA and protein levels of ABCG1 in osteoclasts. Besides, we found that silencing of ABCG1 by ABCG1 siRNA increased the number of osteoclasts, the average diameter of osteoclasts and the fusion index. MiR-216b reduced cholesterol efflux from osteoclasts by inhibiting ABCG1 expression.Conclusion Collectively, these findings suggest that miR-216b downregulates ABCG1 expression and inhibits osteoclast cholesterol efflux, which disturbs cholesterol homeostasis and promotes osteoclastogenesis.