Matrix Vesicles (MVs) budding from osteoblasts, chondroblasts and odontoblasts are accumulated in the ECM and contribute to initial mineralization processes. Growing evidence indicates that MVs share several features residing in exosomes, small membrane vesicles of endosomal origin. Exosomes have gained much attention for their role in intercellular transport of miRNAs that function gene silencing in recipient cells, raising the question of whether MVs include miRNAs. Our findings that MVs isolated from osteoblast cultures suppress RANKL-induced osteoclast formation and bone resorption in vitro prompted us to conduct a global analysis of miRNAs, some of which may reflect the anti-osteoclastogenic activity of MVs. We identified 172 miRNAs in mouse MVs, and 72 were conserved in humans. Among these, we focused on miR-125b, since miR-125b mimicked the anti-osteoclastogenic effect of MVs and accumulated in the ECM of bone. We generated transgenic (Tg) mice expressing miR-125b under the control of the human osteocalcin promoter. Tg mice showed a marked increase in bone volume, especially in trabecular bone, which was due to a decrease in the number of osteoclasts without significant effect on the number of osteoblasts. Calvaria cells and bone marrow macrophages from Tg mice normally differentiated into osteoblasts and osteoclasts, respectively. The anti-osteoclastogenic effect of Tg MVs was larger than that of wild-type MVs. Besides using the 3’UTR targeting reporter, miR-125b-transfected RAW cells showed that Prdm1, a transcription repressor of antiosteoclastogenic factors, was a target of miR-125b. Administration of miR-125b suppressed LPS-induced bone resorption in mice, suggesting that miR-125b represents a potential therapeutic target for osteolytic bone diseases. Thus, our findings demonstrate that miR-125 in MVs accumulates in the ECM during bone formation and acts as a negative regulator of osteoclast formation during bone resortption by targeting Prdm1.