Our previous study using suppression subtractive hybridization (SSH), cDNA microarray and semi-quantitative RT-PCR showed that RPS12 was overexpressed in gastric cancer and it was closely related to metastasis. However, the role of RPS12 in gastric cancer is not clear, which led us to conduct the current study to further investigate the effects of RPS12 on the proliferation and migration of gastric cancer cells, and also to explore the underlying molecular mechanisms. RNA interference was used to inhibit the expression of RPS12. The expression of RPS12 and S100A4 in gastric cancer cells was determined using semi-quantitative RT-PCR and western blot analysis. Cell proliferation and migration were detected by MTT and transwell assay, respectively. In addition, the promoter activity of S100A4 was measured by a Dual-Luciferase Reporter Assay System. We found that RPS12 downregulation led to reduced proliferation and migration of BGC823 and SGC7901 gastric cancer cells. Further results showed that RPS12 inhibition led to reduced S100A4 expression and decreased promoter activity of S100A4 in BGC823 cells. We demonstrated that ectopic expression of S100A4 reversed the reduced proliferation and migration ability after RPS12 inhibition in BGC823 cells. Our findings provide the first demonstration that RPS12 plays important roles in regulating the proliferation and migration of gastric cancer cells. S100A4 can mediate the effects of RPS12 as a downstream effector.