Abstract: Objective To investigate the expression pattern of mitochondrial trifunctional protein subunit HADHA and HADHB in the progression of hilar cholangiocarcinoma and to evaluate its clinical significance in the outcome of cancer patients.MethodsThe tissue microarray was constructed which contains 91 cases of hilar cholangiocarcinoma. Immunohistochemistry was applied to screen the expression of HADHA and HADHB. The expression levels were statistically analyzed to investigate their correlation with clinicopathological features and survival duration of patients. Results The over-expression rates of HADHA and HADHB proteins were 71.4％（65／91） and 59.3％（54／91）， higher than 10.4％（5／48）， 20.8％（10／48）of adjacent normal tissues with statistical significance（P＜0.05）. The expression of HADHA was significantly related with tumor invasion depth and surgical margin （P＜0.05）， and HADHB expression was significantly associated with surgical margin and nodal metastasis （P＜0.05）. The overexpression of HADHA and HADHB could both shorten the overall survival （OS） and progressionfree survival （PFS） of patients. Patients with high HADHA and HADHB expression had a lower PFS and OS than those with low HADHA and HADHB expression， respectively（P＜0.05）. Cox proportional hazard model showed that tumor invasion depth， nodal metastasis， differentiated degree and HADHB were independent prognostic factor for patients with hilar cholangiocarcinoma. Conclusion HADHA and HADHB play remarkably oncogenic roles in the progression of hilar cholangiocarcinoma. HADHB alone is also an independent prognostic factor for cancer patients. HADHA and HADHB can be promising therapeutic targets for the treatment of hilar cholangiocarcinoma in the future.