Homograft rejection is the main cause of heart transplantation failure. The role of TLR2, a major member of the toll-like receptor (TLR) family, in transplantation rejection is has yet to be elucidated. In this study, we used a mouse model of acute cardiac transplantation rejection to investigate whether the TLR2 signalling pathway can regulate cardiac transplantation rejection by regulating alloreactive IL-17+γδT (γδT17) cells. We found that the expression of TLR2 on the surface of dendritic cells (DCs) and macrophages increased during acute transplantation rejection. In addition, our investigation revealed that γδT17 cells exert a significant influence on acute cardiac transplantation rejection. TLR2 gene knockout resulted in an increase in alloreactive γδT17 cells in the spleen and heart grafts of recipient mice compared with wild-type recipient mice and an increase in the mRNA expression of IL-17, IL-1β, CCR6, and CCL20 in the heart grafts. In an in vitro experiment, a mixed lymphocyte reaction was conducted to assess the impact of TLR2 deficiency on the generation of γδT17 cells, which further substantiated a significant increase compared to that in wild-type controls. Furthermore, the mixed lymphocyte reaction showed that TLR2 regulated the production of γδT17 cells by regulating the ability of DCs to secrete IL-1β. These results suggest that TLR2 signalling is important for regulating the generation of γδT17 cells after cardiac allograft transplantation.