MicroRNAs (miRNAs) are a class of noncording RNAs that control gene expression by translational inhibition and messenger RNAs (mRNAs) degradation in plants and animals. Although miRNAs have been implicated in developmental and homeostatic events of vertebrates and invertebrates, the role of miRNAs in bone metabolism has not been explored. Here, we show that microRNA-223 (miR-223) is expressed in RAW264.7 cells, mouse osteoclast precursor cell lines, and plays a critical role in osteoclast differentiation. We constructed miR-223 short interfering RNA (siRNA) or precursor miR-223 (pre-miR-223) overexpression retroviral vectors, and established miR-223 knockdown by siRNA or pre-miR-223 overexpression in stably infected RAW264.7 cells. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were observed in miR-223 knockdown cells as well as control cells. In contrast, pre-miR-223 overexpression completely blocked TRAP-positive multinucleated cell formation compared with control cells. Apoptotic cells were not observed in this study. Our results indicate that miR-223 plays an essential role during osteoclast differentiation, and miR-223 might be a viable therapeutic target for a range of bone metabolic disorders with excess osteoclast activity.