Recognition of evolutionarily conserved ligands by Toll-like receptors (TLRs) triggers signaling cascades in innate immune cells to amplify adaptive immune responses. Nearly all TLRs require MyD88 to transduce downstream signaling. MyD88 deficiency has been shown to promote the allograft acceptance in mice. However, direct evidence for therapeutic potential of MyD88 inhibitors remains lacking. Herein, we used a MyD88 inhibitor, namely ST2825, to explore its therapeutic potential and mechanisms in fully allogeneic skin and heart transplant models. Phenotypic maturation of dendritic cells stimulated by TLR ligands was alleviated by ST2825 in parallel with reduced T-cell proliferation in vitro. A short-course treatment with ST2825 significantly prolonged cardiac graft survival (mean survival time = 18.5 +/- 0.92 days vs. 7.25 +/- 0.46 days). ST2825-treated group had significantly reduced proinflammatory cytokines in allografts compared with control group. ST2825 combined with anti-CD154 induced long-term skin allograft acceptance in about one-third of recipients (>100 days). Skin-tolerant' recipients showed attenuated donor-specific IFN- responses, intact IL-4 responses, and compromised alloantibody responses. We conclude that MyD88 inhibitor ST2825 attenuates acute cardiac rejection and promotes donor-specific hyporesponsiveness in stringent skin transplant models. The direct evidence suggests that pharmacological inhibition of MyD88 hold promising potential for transplant rejection.