OBJECTIVE The male predominance of gastric cancer suggests that female sex hormones may have a protective effect against gastric cancer. We evaluated the expression of estrogen receptors in gastric cancer tissue and cells and the clinical significance of ER-β expression in gastric cancer. METHODS ER-α, ER-β proteins extracted from normal stomach, gastric cancer tissues, and cultured gastric cancer cells (KATO-III, mkn28, mkn45, and mkn74) were assessed by Western blot analysis. The clinical significance of ER-β was explored using tissue microarray methods and immunohistochemical staining of specimens from 148 gastric cancers. RESULTS Both ER-α and β protein expression were noted in normal and gastric cancer tissues. However, in cultured gastric cells, only ER-β was noted in mkn28 and mkn74. Of 148 gastric cancers, 67 (45.3%) were ER-β positive. The ER-β positive group was associated with lower tumor stage, Lauren's intestinal type, negative perineural invasion, and free of recurrence. The ER-β positive group had a better 3-year survival compared with the negative group in survival analysis. CONCLUSIONS Our results suggest that the presence of ER-β in gastric cancer could have a protective effect against invasiveness of gastric cancer. Further studies are needed to clarify the role of ER-β in gastric cancers.