Aims: Cancer stem cells (CSCs) indicate multipotent cells exhibiting self-renewal capacity, multilineage differentiation ability and high carcinogenesis, and are closely related to tumor growth, metastasis, recurrence and chemoresistance. As tumorigenic drivers, CSCs should be effectively targeted to achieve long-lasting therapeutic responses. This study aimed to explore the mechanism of the novel Tripterygium wilfordii Hook F (TWHF) extract Demethylzeylasteral (ZST93) on inhibiting human pancreatic CSCs. Methods: Serum-free floating culture system was used to isolate CSCs. CCK-8 assay was used to evaluate the chemosensitivity. Apoptosis was evaluated by flow cytometry. Autophagy level was evaluated by transmission electron microscopy and immunofluorescence. The activity levels of caspase-3, ERK1/2 and Akt/mTOR pathways were determined by Western blot. Results: Tumorspheres had differentiation ability and stem celllike properties. ZST93 could inhibit the number and diameter of tumorspheres. ZST93 could induce apoptotic cell death in pancreatic CSCs at high concentrations, but not at low concentrations. The apoptosis induced by ZST93 was associated with the significant up-regulation of active caspase-3 expression. ZST93 could induce autophagic cell death in pancreatic CSCs at low concentrations, but not at high concentrations. The autophagy induced by ZST93 was associated with the significant up-regulation of p-ERK1/2 and down-regulation of p-Akt and p-mTOR expression. Conclusions: We revealed that ZST93 inhibits pancreatic CSCs through two different mechanisms, low concentrations of ZST93 could induce autophagic cell death by activating ERK1/2 pathway and inhibiting Akt/mTOR pathway, and high concentrations of ZST93 could induce caspase-3-dependent apoptotic cell death. ZST93 is a potential therapeutic agent for developing novel therapeutic strategies in human pancreatic cancer.