Previously, we used the reverse transcription-polymerase chain reaction (RT-PCR) to show that mammaglobin (MGB1) can serve as a differential marker of breast cancer metastasis from primary lung cancer. However, mRNA-based methods are not appropriate for use in clinical practices. In this study, we examined MGB1 protein expression in 480 tumors from various organs using immunohistochemical detection and a tissue microarray technique. Breast cancers expressing MGB1 were also analyzed clinicopathologically to determine whether these cancers constitute a characteristic subset. Immunohistochemically, MGB1 was expressed specifically in breast cancers. Of the other cancers examined, including 29 of the head and neck, eight of the thyroid, 106 of the lung, 35 of the gastrointestinal tract, three of the pancreas, 14 of the uterine cervix and 13 of the ovary, none were positive for MGB1 except a proportion of salivary gland tumors (6/11, 55%) and endometrial cancers (3/23, 13%). Among the 238 breast cancers, MGB1 was expressed in 114 (48%), most of which were classified histologically as invasive duct or lobular carcinomas. Clinicopathologically, MGB1 expression was associated with positive expression of estrogen receptors and negative expression of CK5, but not with pathological stage, HER2 gene amplification or p53 immunoreactivity. Kaplan-Meier analysis revealed prolonged disease-free survival in patients with MGB1-positive breast cancers (log rank test, P=0.016), but the Cox proportional hazard model failed to confirm that MGB1 was an independent prognostic factor (hazard ratio 1.77, P=0.1755). In terms of practical diagnosis, MGB1 immunohistochemistry can serve as a differential marker of breast cancer metastasis from primary lung cancer for two reasons. Firstly, HER2-positive breast cancer frequently lacks estrogen receptor expression, but MGB1 is expressed in about half of this subtype. Secondly, as primary lung adenocarcinomas may express estrogen receptors, MGB1 expression provides further discrimination of the origin of breast cancers.