Many studies have revealed that S100A4 is involved in cancer progression by affecting a variety of biological functions. Our previous study showed that S100A4 influences many biological properties of gastric cancer cells; however, the underlying mechanisms are far from clear. In this study, we used cDNA microarray analysis to investigate the global alterations in gene expression in MGC803 gastric cancer cells after siRNA-mediated S100A4 inhibition. Among the total genes investigated, 179 differentially expressed genes (38 upregulated and 141 downregulated) were detected in S100A4-siRNA transfected MGC803 cells compared with NC-siRNA transfected cells. We focused on the GDF15 gene, which was significantly downregulated after S100A4 inhibition. ChIP studies showed that the S100A4 protein binds to the GDF15 promoter, implicating S100A4 in GDF15 regulation at the transcriptional level. GDF15 overexpression promoted CSC-like properties of MGC803 cells, such as spheroid and soft-agar colony forming abilities. S100A4 inhibition suppressed the CSC-like properties of the cells, whereas, GV141-GDF15 vector transfection reversed these effects. Our results suggest that S100A4 influences the CSC-like properties of MGC803 gastric cancer cells by regulating GDF15 expression.