Cancer stem cells (CSCs) are the primary source of tumor recurrence and chemoresistance, which complicates tumor treatment and has a significant impact on poor patient prognosis. Therefore, the discovery of inhibitors that specifically target CSCs is warranted. Previous research has established that the TGF-β/Smad signaling pathway is critical for the maintenance of CSCs phenotype, thus facilitating CSCs transformation. In this regard, Celastrus orbiculatus ethyl acetate extract (COE) was shown to exert anticancer properties; however, its therapeutic impact on gastric cancer stem cells (GCSCs) remains unknown. We here demonstrate that COE displayed a strong inhibitory effect on GCSCs growth and CSCs markers. Moreover, COE was shown to efficiently inhibit the development of tumor spheres and accelerate GCSCs apoptosis. Mechanistically, we established that COE could suppress the stemness phenotype of GCSCs by inhibiting the activity of the TGF-β/Smad signaling pathway. To summarize, our data indicate that COE suppresses the malignant biological phenotype of GCSCs via the TGF-β/Smad signaling pathway. These findings shed new light on the anticancer properties of COE and suggest new strategies for the development of efficient GCSCs therapeutics.Keywords: Cancer stem cells; Celastrus orbiculatus extract; Gastric cancer; TGF-β.

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