Background— Interleukin-17 (IL-17), which is predominantly produced by T helper 17 cells distinct from T helper 1 or T helper 2 cells, participates in the pathogenesis of infectious, autoimmune, and allergic disorders. However, the precise role in allograft rejection remains uncertain. In the present study, we investigated the role of IL-17 in acute allograft rejection using IL-17-deficient mice. Methods and Results— Donor hearts from FVB mice were heterotopically transplanted into either C57BL/6J-IL-17-deficient (IL-17 ^{− / −} ) or -wild-type mice. Allograft survival was significantly prolonged in IL-17 ^{− / −} recipient mice due to reduced local inflammation accompanied by decreased inflammatory cell recruitment and cytokine/chemokine expression. IL-17 ^{− / −} recipient mice exhibited decreased IL-6 production and reciprocally enhanced regulatory T cell expansion, suggesting a contribution of regulatory T cells to prolonged allograft survival. Indeed, allografts transplanted into anti-CD25 mAb-treated IL-17 ^{− / −} recipient mice (regulatory T cell-depleted) developed acute rejection similar to wild-type recipient mice. Surprisingly, we found that gamma delta T cells rather than CD4 ⁺ and CD8 ⁺ T cells were key IL-17 producers in the allografts. In support, equivalent allograft rejection was observed in Rag-2 ^−/− recipient mice engrafted with either wild-type or IL-17 ^{− / −} CD4 ⁺ and CD8 ⁺ T cells. Finally, hearts transplanted into gamma delta T cell-deficient mice resulted in decreased allograft rejection compared with wild-type controls. Conclusions— During heart transplantation, (1) IL-17 is crucial for acceleration of acute rejection; (2) IL-17-deficiency enhances regulatory T cell expansion; and (3) gamma delta T cells rather than CD4 ⁺ and CD8 ⁺ T cells are a potential source of IL-17. IL-17 neutralization may provide a potential target for novel therapeutic treatment for cardiac allograft rejection.