Objective:To study the amplification / overexpression of human epidermal growth factor receptor 2 (HER2) in patients with gallbladder cancer, and to analyze the correlation between amplification/overexpression of HER2 with clinicopathological features and survival in patients after R 0 resection. Methods:There were 14 males and 26 females, aged (60.3±8.7) years old and treated at the Cancer Hospital of Chinese Academy of Medical Sciences from January 2011 to December 2016 who met the inclusion criteria of the study. Immunohistochemistry and fluorescence in situ hybridization were used to detect amplification / expression of HER2 in resected tumor tissues. Patients were divided into two groups according to the HER2 gene expression: the HER2-negative group ( n=40) and the HER2-positive group ( n=10). The Chi-square test was used to analyze the relationship between amplification/expression of HER2 and clinicopathological parameters. Patients were followed up by telephone for prognosis. The Kaplan-Meier method was used for survival analysis. The Cox proportional hazard model was used to explore factors affecting prognosis of gallbladder cancer patients. Results:HER2 amplification/overexpression was found in 10 patients with gallbladder cancer, with a positive rate of HER2 being 20.0% (10/50). There was a significant difference in the proportion of patients with vascular tumor thrombus between the HER2 negative group and the HER2 positive group [7.5%(3/40) vs. 30.0%(3/10), P<0.05]. On follow-up, data of 46 patients was available. There were 36 patients in the HER2-negative group and 10 patients in the HER2-positive group. Compared with the HER2-negative group, the median recurrence-free survival (10.10 vs. 75.07 months) and the median overall survival (16.77 vs. 83.07 months) of the HER2-positive group were both significantly lower (both P<0.05) than the HER2-negative group. Univariate analysis showed HER2 positivity to be a risk factor for recurrence-free and overall survival in patients with gallbladder cancer after radical resection. Cox multivariable analysis revealed that lymph node metastasis was an independent risk factor for both recurrence-free ( HR=4.31, 95% CI: 1.92-9.68, P<0.001) and overall survival ( HR=3.44, 95% CI: 1.08-11.00, P=0.037). Conclusion:Amplification / overexpression of HER2 was associated with venous invasion and worse prognosis in patients with gallbladder cancer.