The occurrence and development of osteoclasts can directly affect the severity of bone destruction in middle ear cholesteatoma. At the same time, cell communication between keratinocytes and fibroblasts can stimulate osteoclast differentiation. However, the molecular mechanism of osteoclast differentiation in cholesteatoma is still poorly understood. In this study, we try to isolate the exosomes of keratinocytes from patients with middle ear cholesteatoma, and explore the effects of keratinocyte-derived exosomes (Ker-Exo) on osteoclast differentiation by co-culturing Ker-Exo with fibroblasts and osteoclast precursor cells. As a result, we confirmed that Ker-Exo primed fibroblasts can up-regulate the expression of RANKL and promote osteoclast differentiation. We revealed that the effect of Ker-Exo depened on its miRNA-17 conponent. Analysis confirmed that miRNA-17 was down-regulated in Ker-Exo, and they can increase RANKL level in fibroblasts, thus promoting the differentiation of osteoclasts. In conclusions, we provide evidence that exosomes miRNA-17 secreted by keratinocytes in patients with middle ear cholesteatoma can up-regulate the expression of RANKL in fibroblasts and induce osteoclast differentiation. (C) 2020 Elsevier Inc. All rights reserved.