25-hydroxycholesterol (25-HC) is implicated in many processes, including lipid metabolism and the immune response. However, the role of 25-HC in RANKL-induced osteoclastogenesis remains largely unknown. Our results showed that 25-HC inhibited miR-139-5p expression in mouse bone marrow macrophages (BMMs) cultured in receptor activator of NF-kappa B ligand (RANKL) and monocyte macrophage colony-stimulating factor (M-CSF). Further investigation suggested that 25-HC promoted the expression of nuclear factor of activated T cell cytoplasmic 1 (NFATcl) and Spl, especially in the presence of RANKL and M-CSF. Meanwhile, 25-HC induced nuclear translocation of NFATcl, resulting in the interaction between NFATc1 and Spl that was confirmed by co-immunoprecipitation. Chromatin immunoprecipitation assay indicated that Spl could bind to miR-139-5p promoter, but NFATcl had no binding capacity. Although forming NFATc1/Sp1 complex increased its binding to miR-139-5p promoter, the complex inhibited the transcriptional activity of Spl. Inhibition of NFATcl increase the expression of miR-139-5p, which might be due to the release of free Spl that could bind to the promoter of miR-139-5p. Enforced expression of miR-139-5p impaired osteoclastogenesis induced by co-treatment with 25-HC and RANKL. These results suggested that 25-HC induced the interaction between NFATcl and Spl, reducing the level of free Spl to inhibit miR-139-5p expression and promote osteoclastogenesis. (C) 2017 Elsevier Inc. All rights reserved.