Osteoclasts， bone resorbing cells， derive from monocyte/macrophage cell lineage. Increased osteoclast activity is responsible for bone destruction in diseases such as osteoporosis， periodontitis and rheumatoid arthritis. Transglutaminases (TGs)， protein crosslinking enzymes， were recently found involved in osteoclastogenesis in vivo， however their mechanisms of action have remained unknown. In this study， we have investigated the role of TG activity in osteoclastogenesis in vitro using four TG inhibitors， NC9， Z006， T101， and monodansyl cadaverine. Our results showed that all TG inhibitors were capable of blocking the entire osteoclastogenesis process. The most potent of the inhibitors， NC9 when added to cultures at different phases of osteoclastogenesis， inhibited differentiation， migration， and fusion of pre-osteoclasts as well as resorption activity of mature osteoclasts. Further investigation into the mechanisms revealed that NC9 increased RhoA levels and blocked podosome belt formation suggesting that TG activity regulates actin dynamics in pre-osteoclasts. The inhibitory effect of NC9 on osteoclastogenesis as well as podosome belt formation was completely reversed with a Rho-family inhibitor Exoenzyme C3. Microtubule architecture， acetylation， and detyrosination of α-tubulin were not affected. Finally， we demonstrated that macrophages and osteoclasts expressed mRNA of three TGs:TG1， TG2， and Factor XIII-A which were all differentially regulated in these cells during differentiation. Immunofluoresence microscopic analysis showed that all three enzymes co-localized to podosomes in osteoclasts. Taken together， our data suggests that TG activity regulates differentiation， migration and fusion of osteoclasts via affecting actin dynamics and that this may involve contribution from all three TG enzymes.