Cardiac allograft vasculopathy (CAV) charactered with aberrant remodeling and fibrosis usually leads to the loss of graft after heart transplantation. Our previous work has reported that extracellular high-mobility group box 1 (HMGB1) participated in the CAV progression via promoting inflammatory cells infiltration and immune damage. The aim of this study was to investigate the involvement of HMGB1 in the pathogenesis of CAV/fibrosis and potential mechanisms using a chronic cardiac rejection model in mice. We found high levels of transforming growth factor (TGF)-beta 1 in cardiac allografts after transplantation. Treatment with HMGB1 neutralizing antibody markedly prolonged the allograft survival accompanied by attenuated fibrosis of cardiac allograft, decreased fibroblasts-to-myofibroblasts conversion, and reduced synthesis and release of TGF-beta 1. In addition, recombinant HMGB1 stimulation promoted release of active TGF-beta 1 from cardiac fibroblasts and macrophages in vitro, and subsequent phosphorylation of Smad2 and Smad3 which were downstream of TGF-beta 1 signaling. These data indicate that HMGB1 contributes to the CAV/fibrosis via promoting the activation of TGF-beta 1/Smad signaling. Targeting HMGB1 might become a new therapeutic strategy for inhibiting cardiac allograft fibrosis and dysfunction.