Amiloride is widely used in clinical practice as a diuretic and is known to interact with the epithelial sodium channel and acid-sensing ion channel proteins, as well as Na+/H+ antiporters and Na+/Ca2+ exchangers. The aim of the present study was to examine the effects of amiloride on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and to elucidate the underlying mechanisms in the RAW264.7 murine macrophage cell line. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were counted and the bone resorption area was estimated. In addition the expression levels of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) mRNA and osteoclast-specific genes, including TRAP, matrix metalloproteinase 9, cathepsin K and osteoclast-associated receptor, were examined using reverse transcription-quantitative polymerase chain reaction. The nuclear factor-kappa B (NF-kappa B) and mitogen-activated protein kinase (MAPK) signaling pathways were also investigated using western blotting. The results showed that amiloride significantly reduced the number of TRAP-positive multinucleated cells as well as the bone resorption area. Amiloride also downregulated the expression of NFATc1 mRNA and inhibited the expression of osteoclast-specific genes. A possible underlying mechanism may be that amiloride suppresses the degradation of the inhibitor of NF-kappa B and blocks the activation of c-Jun N-terminal kinase, extracellular signal-regulated kinase and p38, thus implicating the NF-kappa B and MAPK pathway is this process. In conclusion, the current data suggest that amiloride is a strong inhibitor of osteoclast differentiation, indicating a novel indication for amiloride in the treatment of bone-loss-related diseases.