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Wogonoside (Wg), a natural flavonoid, has anticancer effects against several human cancers. The purpose of the present study was to investigate the antitumor effects and underlying mechanisms of Wg on gastric cancer (GC) cell lines. We report that Wg treatment inhibited cell viability and induced apoptosis in human GC cell lines AGS and SGC‐7901, and also retarded GC tumor growth in xenograft mice in vivo. We also found that the Wg exerted its antitumor effects against GC cells via induction of reaction oxygen species accumulation, mitochondrial dysfunction, and endoplasmic reticulum stress. Furthermore, C/EBP homologous protein knockdown inhibited apoptosis and increased the viability of Wg‐treated GC cells. Our findings may facilitate the development of novel therapeutic agents for the treatment of GC.