Members of the HER (ERBB) receptor protein tyrosine kinase family play an important role in regulating cellular division, proliferation, differentiation, and migration and have prognostic significance in a number of cancers. Here, we sought to define their role in extrahepatic cholangiocarcinoma (EHCC). HER2 and HER3 protein expression was studied in 230 EHCC cases using a tissue microarray and compared with clinicopathological variables, including the survival of EHCC patients. HER3 was predominantly localized to the cytoplasm, whereas HER2 exhibited a membranous expression pattern. Overexpression of HER2 and HER3 was observed in 6 % (13/224) and 39 % (90/230) of EHCCs, respectively. Membranous HER2 overexpression occurred more frequently in intraductal papillary neoplasms with an associated invasive carcinoma than in tubular adenocarcinomas (P = 0.02). HER3 protein was more commonly overexpressed in nodular and infiltrative than in papillary tumors (P = 0.03). HER3 overexpression was associated with decreased survival in both univariate (P = 0.01) and multivariate (P = 0.008) analyses, whereas HER2 overexpression was not associated with survival. HER2 and HER3 are overexpressed in subsets of EHCC patients. Notably, HER3 overexpression is correlated with decreased patient survival, suggesting that HER3 constitutes a prognostic factor as well as a potential candidate for targeted therapy.