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Resistance to medications in cancer cells suggests novel methods to increase therapeutic activity. Colorectal cancer is defined as a dangerous malignancy in humans. Amygdalin (AM) is a cytotoxic drug that produces anticancer molecules through its metabolism. Betaglucosidase enzyme (BGL) is the metabolic enzyme of AM and Hydrogen cyanide acid (HCN) is one of the metabolites produced from AM. The purpose of this study was to determine the apoptotic and immunological activity of amygdalin (AM) following activation with the betaglucosidase enzyme (BGL). This case control study was used MTT test to determine the AM toxicity in colorectal cancer cell line (HT-29). Flow cytometry and PI/annexin V labeling were used to determine induced apoptosis. The gene expression of [caspase-3 (CASP-3), apoptosis and caspase activation inhibitor (AVEN), TGFB1-induced anti-apoptotic factor 1 (TIAF-1), and interlukine-1 receptor (IL-1R) genes was determined using real-time PCR. Expression of proteins of the same genes was tested by western blot. AM and AM-BGL had a strong inhibitory effect on cell proliferation when compared to controls. With regards to AM-BGL, a large rise in the mRNA levels of CASP-3 and IL-1R was seen in the treated HT-29 cells, whereas a significant drop in the levels of AVEN-1 and TIAF-1 was observed. CASP-3 and IL-1R protein expression was dramatically increased in treated HT-29, but AVEN-1 and TIAF-1 protein expression was significantly decreased. However, AM-BGL induced significant apoptosis in both early and late phases of cell lines. AM-BGL worked in the direction of cell death by activating the apoptotic pathway and altering the expression of the CASP-3 gene. As a result, AM-BGL may be evaluated as a potential novel anticancer agent.