Abstract Background The purpose of this study was to address the question of how the premature senescence process may affect corneal endothelium after penetrating keratoplasty, because the quality of donor corneal endothelial cells is important for corneal transplant success. Methods The cell senescence and induced oxidative stress in corneal endothelium were assessed using a normal-risk orthotopic mice corneal transplantation model. Senescence associated beta-galactosidase (SA-beta-Gal) staining was used to evaluate premature senescence in the endothelium of corneal allografts. Oxidative Stress and Antioxidant Defense RT2-PCR Arrays and in vitro experimental model using H2O2 treatment were used to investigate the possible mechanism. Results SA-beta-Gal positivity was observed obviously in mice corneal endothelium of allogenic group and the levels of p16INK4a message and protein increased in endothelium of allogenic group compared to syngenic group. By PCR array, an oxidant-antioxidant imbalance was found in the endothelium of corneal allograft after PKP. The results from mice model were validated using human endothelium samples of corneal allograft after PKP. We also developed an in vitro experimental model using H2O2 treatment to simulate a state of oxidative stress in cultured human corneal endothelial cells (HCECs) and found that elevated ROS levels, the up-regulation of CDK inhibitors and ROS-mediated p16INK4A up-regulation in HCECs occur via the ASK1-p38 MAPK pathway. Conclusions Our results demonstrate the presence of oxidative stress and premature senescence in the endothelium of corneal allografts following PKP.