Malignant glioma is a lethal brain tumor that is highly resistant to standard therapy. Our research aims to explore the suppressive effects of nitidine chloride (NC) on gliomas and the mechanisms involved, showing that it is a potential agent for integrative therapy of gliomas. After glioma cells were treated with NC, several experiments were performed to evaluate NC’s antitumor effects. CCK-8 assay was used to detect viability. Transwell and 3-dimensional spheroid invasion assays were used to evaluate motility of glioma in vitro, and the sphere-formation assay showed NC’s influence on glioma stem cells. Apoptosis and intracellular reactive oxygen species were measured by means of flow cytometry. Subcellular structures were observed through transmission electron microscopy. Western blot analysis reflected expression of endoplasmic reticulum (ER) stress and epithelial-mesenchymal transition (EMT) marker proteins. An orthotopic xenograft model was established to investigate the tumor suppressive effects in vivo. Nitidine chloride inhibited glioma cell migration and invasion in vitro, downregulated the EMT proteins, and suppressed sphere formation of glioma stem cells. Furthermore, NC induced persistent ER stress that contributed to apoptosis and reactive oxygen species production. The xenograft model showed that NC effectively restricted glioma growth and invasion in vivo. Furthermore, we confirmed the signaling pathways that ER stress downregulates C/EBPβ and slug, as well as inhibition of the AKT/GSK3β/β-catenin axis caused by NC, in U-87 MG. We demonstrated that NC inhibits gliomas in vitro and in vivo by activating ER stress and downregulating EMT, which provides a basis for glioma therapy.