Eye-derived antigen-presenting cells (APCs) are known to contribute to the immune privilege status of the eye by inducing a form of peripheral tolerance that deviates Th1 type of pro-inflammatory immune responses. Similar systemic tolerance can also be induced by non-ocular APCs exposed to transforming growth factor β (TGFβ) in vitro. Such APCs were found to express enhanced levels of thrombospondin (TSP)-1, an extracellular matrix (ECM) protein. In this report, we analyzed the significance of TSP-1 in conferring tolerance-inducing properties on APCs. While TSP-treated APCs matched TGFβ-treated APCs in their functional ability to induce systemic tolerance, a deficiency of TSP-1 or its receptor CD36 prevented APCs from becoming tolerogenic in response to TGFβ. Exogenous TSP-1 restored tolerogenic ability of TGFβ-treated TSP-1 null APCs. Both TGFβ-treated TSP-1 null and CD36 knockout APCs failed to inhibit IL-12 secretion. Furthermore, TGFβ-treated TSP-1 null APCs, unlike similarly treated wild-type APCs, failed to increase secretion of active TGFβ. Similar to TGFβ, TSP could also up-regulate expression of MIP-2, TGFβ2 and tumor necrosis factor α—all of which are required for tolerance induced by TGFβ-treated APCs. We conclude that TSP-1, an ECM protein induced by TGFβ treatment, orchestrates the changes in APC functional programs that equip these cells to promote tolerance of the eye-derived type.