RUNX3 is a novel tumor suppressor in gastric carcinogenesis and an important factor for differentiation of chief cells in the normal gastric fundic mucosa. In this study, we confirmed RUNX3 immunolocalization in the fundic gland (bottom part) but minimum in surface mucous cell epithelium (top part) in the isolated gland from fundic mucosa. We also analyzed RUNX3 expression by immunohistochemistry in 102 gastric cancers and made a histological assessment of the expression of differentiation markers to evaluate interrelations. Among them, 45 and 57 cases were judged to be RUNX3 positive and negative, respectively, and 33 and 69 cases were pepsinogen I positive and negative, with no link to histological types. RUNX3 expression was significantly associated with that of pepsinogen I (P<0.001), but not mucins, including MUC5AC and MUC6, or the parietal or intestinal phenotypes. In conclusion, the present study showed, for the first time to our knowledge, a relation between RUNX3 and pepsinogen I expression in human gastric cancers. RUNX3 is strongly associated with chief cell phenotypic expression in human gastric cancers, as well as in normal gastric mucosa, and could be considered to play an important role in maintaining the chief cell phenotype.