Hispidulin (4’,5,7-trihydroxy-6-methoxyflavone) is a phenolic flavonoid isolated from the medicinal plant S.involucrata,which exhibits anti-neoplastic activity against several types of cancer.However,the mechanism underlying its anti-cancer activity against hepatocellular carcinoma (HCC) has not been fully elucidated.In this study,we investigated whether and how hispidulin-induced apoptosis of human HCC cells in vitro and in vivo.We showed that hispidulin (10,20 μmol/L) dose-dependently inhibited cell growth and promoted apoptosis through mitochondrial apoptosis pathway in human HCC SMMC7721 cells and Huh7 cells.More importantly,we revealed that its pro-apoptotic effects depended on endoplasmic reticulum stress (ERS) and unfolded protein response (UPR),as pretreatment with salubrinal,a selective ERS inhibitor,or shRNA targeting a UPR protein CHOP effectively abrogated hispidulin-induced cell apoptosis.Furthermore,we showed that hispidulin-induced apoptosis was mediated by activation of AMPK/mTOR signaling pathway as pretreatment with Compound C,an AMPK inhibitor,or AMPK-targeting siRNA reversed the pro-apoptotic effect of hispidulin.In HCC xenograft nude mice,administration of hispidulin (25,50 mg/kg every day,ip,for 27 days) dose-dependently suppressed the tumor growth,accompanied by inducing ERS and apoptosis in tumor tissue.Taken together,our results demonstrate that hispidulin induces ERS-mediated apoptosis in HCC cells via activating the AMPK/mTOR pathway.This study provides new insights into the anti-tumor activity of hispidulin in HCC.