Although p53 gene mutation is a rare event in human nasopharyngeal carcinoma (NPC), over-expressed/accumulated p53 protein is dysfunction in most of NPC. However, till now the mechanism of p53 protein inactivation remains unclear. In order to elucidate the mechanisms of p53 inactivation, p53 interacting proteins in the human NPC HNE1 and HNE2 cells were separeted and identified. p53 binding proteins were recovered by anti-p53 antibody immunoprecipitation with the total proteins from NPC HNE1 and HNE2 cells respectively. The recovered protein complexes were subjected to SDS-PAGE. The 5 separated protein bands were cut from the gel, in-gel digested and analyzed by LC-ESI-MS/MS. Then proteins were identified by peptide sequence tags(PST) and database searching, and were confirmed by immunoprecipitation and Western blot analysis. The results show that nine p53 binding proteins were identified, which were GRP-78 and GRP-75 of HSP 70 family members, GRP-94 of HSP 90 family members, laminA/C, Alpha-actinin 4, Ezrin/Cytovillin, DNA replication licensing factor/MCM3 protein, CD98/4F2 heavy chain and protein kinase C. It can be concluded that this study first time identified nine p53 binding proteins in NPC, which provide important clues to elucidate the mechanism of p53 over-expression and inactivation in NPC.