INTRODUCTION: Shortage of donor organs is one of the greatest challenges of cardiac transplantation. Xenotransplantation is a potential way to solve the contradiction of imbalance and pigs are considered ideal donor sources. However, xenotransplantation still faces the problem of immune rejection at present. In order to further understand the molecular picture of immune rejection after xenotransplantation, and develop immunosuppressive agents to further overcome rejection, we conducted a proteomic analysis of a heterotopic pig-to-non-human primate (NHP) animal model. METHODS: We constructed a heterotopic NHP animal model using wild-type (WT) and alpha-1,3-galactosyltransferase gene knockout (GTKO) porcine hearts as donors. Based on quantitative proteomics analysis, we investigated the changes of protein after CXTx in three groups: Group I: WT donor heart, Group II: GTKO donor heart without immunosuppression, and Group III: GTKO donor heart with immunosuppression. Finally, we assessed the efficacy of the target using a heterotopic heart transplantation model from SD rats to Balb/c mice. RESULTS: A total of 2425 proteins were identified in the donor heart tissues and approximately 15% of proteins were significantly changed after CXTx, most of them had increased expression. The results of proteomic analysis demonstrated that chronic hypoxia injury induced by microvascular thrombosis may play an important role during cardiac xenograft failure, confirmed by histopathological results. Remarkably, we showed some novel targets especially increased expression of pentraxin 3, MVP, and HSP90AB1 that cannot be suppressed in the present gene editing and immunosuppressive interventions. Because NF-κB is a common downstream regulator of these three proteins, we hypothesize that it may be crucial to the occurrence of xenograft failure and considered as a potential therapeutic target. Using the SD Rat-Balb/C Mouse CXTx model and inhibiting NF-κB with BAY 11-7082, we found that NF-κB targeting prolonged graft survival from 5 to 8 days and reduced myocardial inflammation. CONCLUSIONS: In summary, the proteomic analysis could help us to solve the mystery of cardiac xenograft failure, confirm the key pathways, and reveal a clear vision of future interventions. NF-κB inhibition effectively decreased immune cell infiltration and antibody deposition in myocardial tissue, suggesting its potential as a therapeutic strategy to enhance graft survival and reduce inflammation in cardiac xenotransplantation (CXTx).