GAGE12I is a tumor metastasis-promoting factor, which can induce gastric cancer cells to invade and migrate. We investigated the effect of miR-28-5p targeting GAGE12I on proliferation, invasion, and migration of human gastric cancer cell lines SGC-7901, AGS, and MGC-803. The expression levels of miR-28-5p and GAGE12I were detected by real-time PCR and western blot, respectively. Cell proliferation, migration, and invasion were measured by MTT and Transwell chamber. The interaction between miR-28-5p and GAGE12I was investigated by bioinformatics analysis and luciferase assay. Results showed that the expression of miR-28-5p in human gastric cancer cell lines was lower than that in normal gastric epithelial cells (
P
<
0.05
). Overexpression of miR-28-5p suppressed cell proliferation, invasion, and migration (
P
<
0.05
). GAGE12I was confirmed as a target of miR-28-5p. Cell proliferation, invasion, and migration were decreased in cells transfected with shGAGE12I compared with those of the scrambled group (
P
<
0.05
). Collectively, miR-28-5p negatively regulated GAGE12I and reduced the proliferation, invasion, and migration of gastric cancer cells.