Gallbladder carcinoma is a relatively rare malignancy with an extremely poor patient prognosis. This malignancy occurs more frequently in women than men, yet expression of the estrogen receptor (ER) family has not been studied. We applied an immunohistochemical technique to examine the expression of ERalpha and ERbeta in gallbladder carcinoma tissues and then compared their expression status with several clinicopathological factors. We examined 26 gallbladder adenocarcinoma specimens and 11 non-cancerous regions using ERalpha and ERbeta antibodies. Expression of the ER isoforms in the cancer specimens was further evaluated at the central area and at the invasive front. ERbeta was expressed in most specimens including cancer and normal region, however ERbeta expression significantly differed between non-cancerous and cancerous regions. Moreover, negative expression of ERbeta at the invasive front was significantly associated with malignant properties of the primary tumor such as lymph node metastasis, advanced stage, lower differentiation of histologic type, lymphatic invasion and a poor prognosis of the patients. In conclusion, evaluation of ERbeta expression at cancer front may be an important factor in identifying a highly malignant group of gallbladder carcinoma.