Objective: To investigate the expression of targeting protein for xenopus kinesin-like protein 2 (TPX2) in cholangiocarcinoma tissue and its clinical significance. Methods: The cancer tissues together with their adjacent tissues from 60 patients with cholangiocarcinoma were collected. The positive expression rates of TPX2 protein in these tissues were determined by immunohistochemical staining, and the TPX2 protein expression levels were detected by Western blot in 4 pairs of randomly selected tissues. The relations of TPX2 protein expression with the clinical factors and prognosis of the patients were analyzed. Results: In cholangiocarcinoma tissue compared with adjacent tissue, the positive expression rate (46.7% vs. 8.3%, P<0.05) and expression level of TPX2 protein were increased. The TPX2 protein expression was significantly associated with the TNM stage, lymph node metastasis and survival of the patients (all P<0.05). The postoperative survival rates in patients with positive TPX2 protein expression were significantly lower than those in patients with its negative expression (2-year survival rate: 27.6% vs. 78.4%; 3-year survival rate: 14.3% vs. 59.5%, both P<0.05). Conclusion: High TPX2 expression may possibly be associated with postoperative recurrence and metastasis in cholangiocarcinoma patients. It may be a potential biological indicator for the early diagnosis and estimation of prognosis and a therapeutic target of cholangiocarcinoma.