The Notch pathway is an important intercellular signaling pathway that plays a major role in controlling cell fate. Accumulating evidence indicates that Notch and its ligands present on antigen‐presenting cells might be important mediators of T helper cell differentiation. In this study, we investigated the role of Jagged2 in murine cardiac transplantation by using a signaling Jagged2 mAb (Jag2) that activates recombinant signal‐binding protein‐Jκ. While administration of Jag2 mAb had little effect on graft survival in the fully allogeneic mismatched model BALB/c→B6, it hastened rejection in CD28‐deficient recipients. Similarly, Jag2 precipitated rejection in the bm12→B6 model. In this MHC class II‐mismatched model, allografts spontaneously survive for >56 days due to the emergence of Treg cells that inhibit the expansion of alloreactive T cells. The accelerated rejection was associated with upregulation of Th2 cytokines and proinflammatory cytokine IL‐6, despite expansion of Treg cells. Incubation of Treg cells with recombinant IL‐6 abrogated their inhibitory effects in vitro. Furthermore, neutralization of IL‐6 in vivo protected Jag2‐treated recipients from rejection and Jagged2 signaling was unable to further accelerate rejection in the absence of Treg cells. Our findings therefore suggest that Jagged2 signaling can affect graft acceptance by upregulation of IL‐6 and consequent resistance to Treg‐cell suppression.