CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) are involved in several autoimmune diseases, including rheumatoid arthritis. TNF-α blockers induce therapeutic benefits in rheumatoid arthritis via a variety of mechanisms. We aimed to characterize the impact on Treg of TNF-α overexpression in vivo and of TNF-α inhibiting treatments. We used human TNF-α transgenic mice as a model of strictly TNF-α-dependent arthritis. Our study showed that initial Treg frequency was lower in TNF-α transgenic mice than in wild-type mice. However, the course of arthritis was marked by elevation of Treg frequency and a dramatic increase in expression of TNFR2. Antagonizing TNF-α with either the anti-human TNF-α Ab (infliximab) or active immunotherapy (TNF-kinoid) increased the Treg frequency and upregulated CTLA-4, leading to enhancement of suppressor activity. Moreover, both anti-TNF-α strategies promoted the differentiation of a CD62L(-) Treg population. In conclusion, in an in vivo model of TNF-α-driven arthritis, Treg frequency increased with inflammation but failed to control the inflammatory process. Both passive and active TNF-α-inhibiting strategies restored the suppressor activity of Treg and induced the differentiation of a CD62L(-) Treg population.