The purposes of this study were to determine the expression profile of Raf kinase inhibitor protein (RKIP) in human gastric cancer cells and its effect on the biological characteristics of SGC-7901 cell lines, to examine the modulatory effect of microRNA-224 (miR-224) on RKIP. The research will provide novel strategies for gastric cancer treatment in the future. Quantitative real-time reverse transcription PCR (qRT-PCR) was employed to determine the expression profile of RKIP in gastric cancer cell lines (SGC-7901, MGC80-3, and MKN45). A eukaryotic expression vector, pcDNA3.1-RKIP, was constructed and transfected into SGC-7901 cells. Changes in RKIP protein expression were examined by Western blot assays, and the effect of RKIP overexpression on SCG-7901 cell viability was determined by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) assays. The effect of RKIP overexpression on SGC-7901 cell proliferation and apoptosis was analyzed by flow cytometry and that on the migration of SGC-7901 cells was investigated by Transwell migration assays. RKIP was identified to be a regulatory target gene of miR-224 using a luciferase reporter gene system, and the effect of miR-224 on intracellular RKIP protein expression was examined by Western blot assays. The regulatory effect of miR-224 on the biological characteristics of RKIP was investigated by MTT, flow cytometry, and Transwell invasion chamber assays. The expression of RKIP in gastric cancer cells was decreased significantly in comparison to that of normal gastric mucosal epithelial cells (GES-1) (p < 0.01), as demonstrated by qRT-PCR assays. Compared with the control group, the up-regulation of RKIP intracellular expression was observed in SGC-7901 cells after transfection of pcDNA3.1-RKIP for 48 h (p < 0.01). There were significant decreases in cell viability and the S-phase fraction (p < 0.05), concomitant with a significant increase in apoptosis (p < 0.01), as well as a significant reduction in cells migrating through Transwell chambers (p < 0.05), as shown by MTT, flow cytometry, and Transwell invasion chamber assays. A significant decrease in luciferase activities in cells transfected with a miR-224 mimic was observed compared with that of the control group (p < 0.05), as suggested by the luciferase reporter gene system. As shown by Western blot assays, there was a significant decrease in RKIP expression in SGC-7901 cells transfected with the miR-224 mimic for 48 h compared with the control group (p < 0.05). As shown by MTT, flow cytometry, and Transwell invasion chamber assays, the changes in biological characteristics induced by RKIP overexpression could be suppressed in SGC-7901 cells after transfection of the miR-224 mimic. In conclusion, the down-regulation of RKIP expression was observed in human gastric cell lines, and miR-224 could negatively regulate the expression and biological characteristics of RKIP, contributing to suppress the proliferation and invasion of gastric cells.