SummaryAn important role of transforming growth factor‐β (TGF‐β) in the development of regulatory T cells is well established. Although integrin‐mediated activation of latent TGF‐β1 is considered essential for the induction of regulatory T (Treg) cells by antigen‐presenting cells (APCs), such an activation mechanism is not applicable to the TGF‐β2 isoform, which lacks an integrin‐binding RGD sequence in its latency‐associated peptide. Mucosal and ocular tissues harbour TGF‐β2‐expressing APCs involved in Treg induction. The mechanisms that regulate TGF‐β activation in such APCs remain unclear. In this study, we demonstrate that murine APCs exposed to TGF‐β2 in the environment predominantly increase expression of TGF‐β2. Such predominantly TGF‐β2‐expressing APCs use thrombospondin‐1 (TSP‐1) as an integrin‐independent mechanism to activate their newly synthesized latent TGF‐β2 to induce Foxp3+ Treg cells both in vitro and in vivo. Expression of Treg induction by TGF‐β2‐expressing APCs is supported by a TSP‐1 receptor, CD36, which facilitates activation of latent TGF‐β during antigen presentation. Our results suggest that APC‐derived TSP‐1 is essential for the development of an adaptive regulatory immune response induced by TGF‐β2‐expressing APCs similar to those located at mucosal and ocular sites. These findings introduce the integrin‐independent mechanism of TGF‐β activation as an integral part of peripheral immune tolerance associated with TGF‐β2‐expressing tissues.