Alpha-Momorcharin (α-MMC), as one of the most important type I RIPs, has been reported to exert inhibitory effects against various tumour cells through its N-glycosidase activity. The present study was designed to propose an efficient purification strategy and explored its mechanism of apoptosis signalling pathway against human liver cancer cells SK-Hep-1. α-MMC can be successfully obtained by our purification strategy combining ion-exchange and gel-filtration chromatography. The functional studies revealed that α-MMC obviously increased the level of ROS and apoptosis rate, induced cell cycle arrest in the G1 phase, and depolarised MMP of SK-Hep-1 cells. To further confirm whether α-MMC could induce mitochondria involved apoptosis, we found that PARP-1, Caspase-3, Caspase-9, and BCL-2 were downregulated upon α-MMC. Taken together, these results suggested that this natural purified α-MMC can induce apoptosis involved mitochondria and may serve as a potential novel therapeutic drug in the treatment of human liver cancer in the future.