Gastric cancer stem cells (GCSCs) have an important role in metastasis and recurrence of gastric cancer, and novel treatment strategies that target GCSCs are urgently required. Although evodiamine (Evo), a derivative of the traditional herbal medicine Evodia rutaecarpa, has been reported to have various biological effects, its effect on GCSCs remains unknown. In order to determine the effect of Evo on apoptosis of GCSCs, an MTS assay, flow cytometry and western blot analysis were performed. The effect of Evo on self‑renewal in GCSCs was measured by alterations in the sphere formation ability, the expression of induced‑pluripotent stem cell factors, expression of epithelial-to-mesenchymal transition (EMT) factors and oxaliplatin resistance of gastric cancer cells (GCCs). Evo inhibited proliferation, promoted the Bax/B‑cell lymphoma 2 ratio and altered active caspase‑3 expression of GCSCs. In addition, Evo decreased the sphere formation ability, the expression of Sox2, KLF4, Bmi‑1 and Oct4, and oxaliplatin resistance in GCCs. Evo decreased the expression of Slug, Twist, Zeb1 and vimentin, suggesting an inhibitory effect on EMT. Furthermore, the expression of β‑catenin, c‑Myc and cyclin D1 was decreased in Evo‑treated spheroids from GCCs. In conclusion, Evo inhibited the Wnt/β‑catenin signaling pathway to inhibit proliferation and stem cell properties of GCSCs and repressed the EMT. The present findings highlight the prospect of Evo as a CSCs-targeted therapy in gastric cancer.