Postmenopausal osteoporosis (PMOP) is a frequent bone disorder responsible for an increased risk of disability to millions of individuals in the world. For identifying novel and effective targets to treat this disease, it is essential to explore the underlying molecular mechanisms. MicroRNAs (miRNAs) have been widely investigated due to their involvement in the pathophysiology of bone loss. In this study, we attempted to elucidate the role of miR-301-b in murine osteoclastogenesis. We found that miR-301-b expression was increased in the bone tissues from PMOP patients, along with up-regulated nuclear factor of activated T cells cl (NFATC1), which were confirmed in ovariectomy (OVX)-induced mouse bone specimens and bone marrow-derived macrophages (BMMs). Osteoclastogenesis was found to be obviously suppressed by miR-301-b inhibitor, whereas being further promoted in BMMs transfected with miR-301-b mimic. The animal studies showed that osteoclastic miR-301-b knockout markedly upregulated the bone mass by reducing osteoclastogenesis. Mechanistically, we found that cylindromatosis (CYLD) was a direct target of miR-301-b at the post-transcriptional level during osteoclastogenesis. The enhanced expression of CYLD led to a reduction of phosphorylated nuclear factor kappa B (NF-kappa B), along with remarkably decreased tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). Finally, osteoclastic miR-301-b ablation evidently inhibited OVX-induced osteoclastogenesis, exhibiting protective effects against bone loss in rodent animals. Therefore, results in the study reported an important mechanism for osteoclastogenesis progression regulated by miR-301-b/CYLD/NF-kappa B pathway, which may be an effective therapeutic target for PMOP treatment. (C) 2020 Published by Elsevier Inc.