Objective: To investigate the effect of genetic alteration(homozygous deletion and point mutation)and expression of p16 gene and p16 protein on hilar cholangiocarcinoma(HCGC) . Methods:Genetic alteration and expression of p16 protein were detected by polymerase chain reaction single strand comformation polymorphism (PCRSSCP) and immunohistochemical method in 36 HCGC tissues. Results:p16 gene revealed alteration in 21 of 36 HCGC tissues (58.3%),among which 8 had homozygous deletion and 13 had point mutation. In HCGC tissues, 8 revealed no p16 protein expression and 10 showed low level expression of p16 protein. Conclusions :The alteration of p16 gene and abnormal expression of p16 protein are significantly correlated with the biological behavior and clinical staging of HCGC,and may be helpful to evalute the malignant degree of HCGC and the patients prognosis.