Objectives: Rheumatoid arthritis (RA) patients have defective CD4+CD25+ regulatory T (Treg) cells and increased osteoclastogenesis. A similar situation has been described in collagen-induced arthritis (CIA). We investigated whether a single transfer of polyclonally activated Treg cells inhibits CIA and osteoclastogenesis. Methods: Purified Treg cells were expanded in vitro with anti-CD3- and anti-CD28-coated beads and injected in DBA/1 mice. Mice were immunized with collagen type II (CII) in complete Freund’s adjuvant (CFA) and scores of arthritis were recorded. In vitro osteoclastogenesis assays were performed on splenocytes by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-[kappa]B ligand (RANKL). Levels of anti-CII antibody and cytokines were determined in the supernatant using ELISA and Bio-Plex protein array system. Results: 106 activated Treg cells significantly counteracted the development of CIA, which was accompanied by decreased serum levels of TNF-[alpha] and IL-6, but not by inhibition of autoimmune antibody responses. The differentiation of osteoclasts in splenocyte cultures was significantly reduced in the presence of prestimulated Treg cells. Expression of cytokines that are described to inhibit osteoclastogenesis, including granulocyte macrophage colony-stimulating factor (GM-CSF), IFN-[gamma], IL-5 and IL-10, were dramatically increased upon addition of Treg cells. Furthermore, splenocytes from mice that had been treated with Treg cells displayed an impaired capacity to develop into mature osteoclasts, suggesting that Treg cells abrogated osteoclastogenesis in vivo. Conclusions. Activated CD4+CD25+ Treg cells improve clinical symptoms of CIA, regulate cytokine production and inhibit osteoclastogenesis in vitro and in vivo.