Objective: RORα is a member of the nuclear receptor superfamily that plays a critical role in cancer. We previously show that upregulation of RORα in gastric cancer cells induced by diallyl disulfide (DADS) were identified using proteomic technique. This study to investigate the effect of RORα in proliferation, migration, and invasion of gastric cancer cells induced by DADS. Methods: The expressions of RORα were detected by Indirect immunofluorescence, RT-PCR and Western blot in gastric cancer cells by DADS. The effect of overexpression and knockdown of RORα on proliferation, migration and invasion in gastric cancer cells by DADS were detected by MTT, soft agar colony formation, flow cytometry, wound healing and Transwell invasion assays. Results: Indirect immunofluorescence showed that the expression rate and fluorescence intensity of RORα in gastric cancer MGC803, BGC823, SGC7901 and MKN28 cells treated by DADS were elevated. RT-PCR and Western Blot showed that the RORα expression was increased. Overexpression of RORα or DADS inhibited the proliferation, migration and invasion in MGC803 cells. However, knockdown of RORα strengthened the capacity of proliferation, migration and invasion. DADS inhibited the migration and invasion in knockdown of RORα cells. However, knockdown of RORα weakened the inhibition of proliferation, migration and invasion induced by DADS in MGC803 cells. Conclusion: DADS can upregulate the expression of RORα in gastric cancer cells. DADS or overexpression of RORα may inhibit the proliferation, migration and invasion in gastric cancer cells. This information suggests that DADS can become a active drug of treatment of gastric cancer, and RORα is a potent tumor suppressor and a potential therapeutic target for gastric cancer.