Osteoblasts and osteoclasts are responsible for the formation and resorption of bone, respectively. An imbalance between these two processes results in a disease called osteoporosis, in which a decreased level of bone strength increases the risk of a bone fracture. MicroRNAs (miRNAs) are small non-coding RNA molecules of 18-25 nucleotides that have been previously shown to control bone metabolism by regulating osteoblast and osteoclast differentiation. In this study, we detected the expression pattern of 10 miRNAs in serum samples from patients with osteoporosis, and identified the altered expression of 6 miRNAs by comparison with patients without osteoporosis. We selected miR-144-3p for further investigation, and showed that it regulates osteoclastogenesis by targeting RANK, and that it is conserved amongst vertebrates. Disrupted expression of miR-144-3p in CD14+ peripheral blood mononuclear cells changed TRAP activity and the osteoclast-specific genes TRAP, cathepsin K (CISK), and NFATC. TRAP staining, CCK-8, and flow cytometry analyses revealed that miR-144-3p also affects osteoclast formation, proliferation, and apoptosis. Together, these results indicate that miR-144-3p critically mediates bone homeostasis, and thus, represents a promising novel therapeutic candidate for the treatment of this disease.