Objective: To investigate the effect of diallyl disulfide (DADS) on NADPH oxidase activity in gastric cancer cells and the mechanism. Methods: In gastric cancer AGS cells after exposure to DADS, the NADPH oxidase activity and miR-34a expression as well as the status of Src-Gab1-Shp2 pathway in the cells were detected. Meanwhile, several interventions were made to investigate the relationship among NADPH oxidase, miR-34a, and Src-Gab1-Shp2 pathway. Results: The NADPH oxidase activity and miR-34a in AGS cells were significantly increased after DADS treatment (both P<0.05), and the NADPH oxidase activity increasing effect of DADS was abolished by Src inhibitor PP2 addition. The mRNA expressions of Src, Gab1 and Shp2 were all significantly decreased after DADS or miR-34a mimics treatment, moreover, the expression levels of phosphorylated Src, Gab1 and Shp2 proteins were all significantly down-regulated after DADS treatment (all P<0.05). Conclusion: DADS can increase NADPH oxidase activity in gastric cancer cells, and this effect may probably be associated with its increasing miR-34a expression and thereby, inhibiting the activity of Src-Gab1-Shp2 pathway.