Our aim was to examine the impact of TLR5 ligation in rheumatoid arthritis (RA) and experimental arthritis pathology. Studies were conducted to investigate the role of TLR5 ligation on RA and mouse myeloid cell chemotaxis or osteoclast formation, and in addition, to uncover the significance of TNF-alpha function in TLR5-mediated pathogenesis. Next, the in vivo mechanism of action was determined in collagen-induced arthritis (CIA) and local joint TLR5 ligation models. Last, to evaluate the importance of TLR5 function in RA, we used anti-TLR5 Ab therapy in CIA mice. We show that TLR5 agonist, flagellin, can promote monocyte infiltration and osteoclast maturation directly through myeloid TLR5 ligation and indirectly via TNF-alpha production from RA and mouse cells. These two identified TLR5 functions are potentiated by TNF-alpha, because inhibition of both pathways can more strongly impair RA synovial fluid-driven monocyte migration and osteoclast differentiation compared with each factor alone. In preclinical studies, flagellin postonset treatment in CIA and local TLR5 ligation in vivo provoke homing and osteoclastic development of myeloid cells, which are associated with the TNF-alpha cascade. Conversely, CIA joint inflammation and bone erosion are alleviated when TLR5 function is blocked. We found that TLR5 and TNF-alpha pathways are interconnected, because TNF-alpha is produced by TLR5 ligation in RA myeloid cells, and anti-TNF-alpha therapy can markedly suppress TLR5 expression in RA monocytes. Our novel findings demonstrate that a direct and an indirect mechanism are involved in TLR5-driven RA inflammation and bone destruction.