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α-Mangostin, a xanthone derivative from the pericarp of mangosteen, exerts both anti-proliferative and cytotoxic effects on various cancers. However, the effect of α-mangostin on hepatocellular carcinoma (HCC) cells has not yet been fully determined. Therefore, the present study investigated the anti-proliferative effect of α-mangostin on HCC cells. α-Mangostin inhibited the proliferation of HCC and malignantly transformed hepatic cells. In addition, α-mangostin induced G0/G1 phase cell cycle arrest. Furthermore, α-mangostin also reversed doxorubicin-resistance in the HCC cells induced by IL-6. Moreover, the percentage of CD133, CD44 and EpCAM positive stem-like HCC cells treated with α-mangostin was reduced. The expression of key stemness transcription factors, such as Bmi-1, c-Myc and Nanog, were also suppressed following α-mangostin treatment. In conclusion, α-mangostin has been shown to have anti-proliferative effects in HCC cells and malignantly transformed hepatic cells. This work discovered the potential effect of α-mangostin in HCC prevention and treatment.