The purpose of this study was to analyze Raf kinase inhibitor protein (RKIP) expression in gastric cancer tissue, its correlation with gastric cancer clinical pathology, and its role in gastric cancer invasion and metastasis in order to provide experimental evidence for the potential biological therapy of this disease. Both immunohistochemistry and western blot analyses were used to test for RKIP expression in 55 cases of gastric cancer tissue and the adjacent gastric mucous membrane tissue. The correlations of RKIP expression with the onset, development, and clinical pathology of gastric cancer were analyzed. After transiently transfecting the human gastric cancer cell line MKN45 with a eukaryotic expression vector containing the full length RKIP cDNA, the changes in MKN45 cell invasiveness and metastatic ability were studied. Immunohistochemistry and western blot results revealed that the quantity of RKIP protein expressed in the gastric cancer tissues was significantly lower than that of the adjacent normal gastric mucous membrane tissues (p < 0.05). The quantity of RKIP protein expression was reduced (p < 0.05) as the gastric cancer cells' differentiation decreased, the TNM stage increased, and the extent of invasion expanded. However, the expression of RKIP in the gastric cancer tissues was not associated with the patients' age or gender (p > 0.05). By overexpressing RKIP in the human gastric cancer cell line MKN45 and through the use of a Transwell invasion chamber, we determined that RKIP overexpression significantly reduced both the invasiveness and metastatic ability of MKN45 cells (p < 0.05). Low or absent RKIP expression may be associated with the onset and development of gastric cancer and its ability to invade and metastasize.