Background: Hepatocellular carcinoma (HCC) remains an aggressive malignancy with a poor prognosis and a leading cause of cancer-related mortality globally. Cumulative evidence suggests critical roles for endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in chronic liver diseases. However, the role of ER stress in HCC pathogenesis, aggressiveness and therapy response remains unclear and understudied. Objectives: Against this background, the present study evaluated the therapeutic efficacy and feasibility of notopterol (NOT), a furanocoumarin and principal component ofNotopterygium incisum, in the modulation of ER stress and cancer stemness, and the subsequent effect on liver oncogenicity. Methods: An array of biomolecular methods including Western blot, drug cytotoxicity, cell motility, immunofluorescence, colony and tumorsphere formation, flow-cytometric mitochondrial function, GSH/GSSG ratio, and tumor xenograft ex vivo assays were used in the study. Results: Herein, we demonstrated that NOT significantly suppresses the viability, migration, and invasion capacity of the human HCC HepJ5 and Mahlavu cell lines by disrupting ATF4 expression, inhibiting JAK2 activation, and downregulating the GPX1 and SOD1 expression in vitro. NOT also markedly suppressed the expression of vimentin (VIM), snail, b-catenin, andN-cadherin in the HCC cells, dose-dependently. Treatment with NOT significantly attenuated cancer stem cells (CSCs)-like phenotypes, namely colony and tumorsphere formation, with the concomitant downregulation of stemness markers OCT4, SOX2, CD133, and upregulated PARP-1 cleavage, dose-dependently. We also demonstrated that NOT anticancer activity was strongly associated with increased cellular reactive oxidative stress (ROS) but, conversely, reduced mitochondrial membrane potential and function in the HepJ5 and Mahlavu cells in vitro. Our tumor xenograft studies showed that compared with sorafenib, NOT elicited greater tumor growth suppression without adverse changes in mice body weights. Compared with the untreated control and sorafenib-treated mice, NOT-treated mice exhibited markedly greater apoptosis ex vivo, and this was associated with the co-suppression of stemness and drug-resistance markers OCT4, SOX2, ALDH1, and the upregulation of endoplasmic reticulum stress and oxidative stress factors PERK and CHOP. Conclusions: In summary, we demonstrated for the first time that NOT exhibits strong anticancer activity via the suppression of cancer stemness, enhanced endoplasmic reticulum stress and increased oxidative stress thus projecting NOT as a potentially effective therapeutic agent against HCC.