Cell-cell fusion is a critical step in osteoclast development, because only after osteoclasts become multinucleated can they efficiently resorb bone. Although recent studies suggest that dendritic cell-specific transmembrane protein (DC-STAMP) may play a key role in the process of fusion of mouse osteoclasts, little information has been available on the role of DC-STAMP in human osteoclasts. In this study, we screened and identified an in vitro-transcribed short-hairpin RNA targeting human DC-STAMP from four candidates and generated a lentivirus vector. Subsequent experiments indicated that this lentiviral transgenic system could effectively transfer into target human osteoclasts, at more than 80 % gene transfer efficiency at multiplicity of infection of 15, and significantly and specifically inhibited DC-STAMP expression at both mRNA and protein levels. We also found that DC-STAMP inhibition by RNAi consequently suppressed fusion and bone resorption of human osteoclasts. In conclusion, these data indicated the lentivirus-mediated RNAi was capable of efficiently suppressing DC-STAMP expression in primary human osteoclasts and inhibiting osteoclastogenesis, demonstrating an essential role of DC-STAMP in the differentiation of human osteoclasts.