Objective: To study the methylation status of several genes on p53-Bax mitochondrial apoptosis pathway and clinical significance in cholangiocarcinoma. Methods: Promoter hypermethylation of DAPK, p14 and ASC genes were de-tected by methylation-specific PCR. p53 gene status (exon 5-8) were examined by automated sequencing, combined with the clinical documents of patients by statistics analysis. Results: (1) We found 66.7% of 36 cases cholangiocarcinoma had meth-ylation of at least one tumor suppressor gene. The frequency of tumor suppressor gene methylation in cholangiocarcinoma was: p14 (24%), DAPK (30.6%), TMSI/ASC (36.1%). The frequency of tumor suppressor gene methylation in tissues near cancer was: DAPK (5.6%), TMSI/ASC (8.3%). (2) p53 gene mutations were found in 22 of 36 patients (61.1%). (3) There were no statistically relationship among the methylation of DAPK, p14 and ASC genes. There were negative relationship differences between the methylation of p14 and p53 gene mutation (P<0.05). (4) p53 gene mutation combined with the methylation of tumor suppressor were 14 cases (38.9%). There were statistically differences on extent of pathologic biology, differentiation and invasion (P<0.05). Conclusion: Our study indicated that methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma was a common epigenetic event. Although the methylation of ASC, DAPK genes was low, it might be significance for early diagnosis, p53 gene mutation combined with the methylation of tumor suppressor might be relationship with pathologic biology, it trended to more malignancy.