The effects of the interaction between KLF4 and β-catenin may be significant in human carcinogenesis and tumor development. This study aimed to determine whether the expression of KLF4 and β-catenin in gastric cancer tissues is associated with clinicopathological characteristics. Western blot analysis and immunohistochemistry were performed to detect KLF4 and β-catenin expression in tumor and corresponding non-cancerous tissues from 49 patients. The data revealed that KLF4 expression was significantly reduced in gastric cancer tissues compared with normal tissues. By contrast, the expression of the β-catenin protein was significantly increased in all tumor tissues, but was not expressed in distant normal mucosae. The altered expression of the KLF4 and β-catenin proteins was associated with advanced tumor stage and gastric cancer. In addition, the expression of the KLF4 and β-catenin proteins was inversely associated in moderately differentiated human gastric cancers. This study showed that β-catenin expression is significantly increased and KLF4 protein expression is markedly decreased in gastric cancer tissues, thus showing that the expression of KLF4 is inversely correlated with that of β-catenin in gastric cancer. The altered expression of the two proteins is associated with advanced tumor stage in gastric cancer.