Th cells that produce IL-17 (Th17 cells) are a distinct subset of Th cells implicated in several autoimmune diseases. Although CD28–B7 interaction has been shown to be involved in Th17 differentiation in vitro, the role of CTLA-4 in controlling Th17 development is completely unknown. We report in this paper that blocking the CTLA-4–B7 interaction potentiates Th17 cell differentiation in vitro and in vivo. Furthermore, blocking CTLA-4–B7 interaction in vivo confers the susceptibility of experimental autoimmune myocarditis to CD28−/− mice or increases the severity of experimental autoimmune myocarditis in wild-type mice. The enhanced disease susceptibility is mediated by heightened Th17 responses. With these results, we are the first to demonstrate that CTLA-4–B7 interaction inhibits Th17 differentiation in vitro and in vivo and suppresses Th17-mediated autoimmunity.