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L-amino acid oxidase isolated from Crotalus mitchellii pyrrhus (Cmp-LAAO) exhibited cytotoxicity against LNCaP prostate adenocarcinoma cells. The viability of LNCaP cells decreased in a concentration- and time-dependent manner upon administration of Cmp-LAAO. Cmp-LAAO induced apoptosis as evidenced by AnnexinV/PI staining using flow cytometry. An increase in caspase-9 and caspase-3 activity were also observed. The damaging effect of LAAO appears to be due to its enzymatic activity, that produces hydrogen peroxide which can then induce oxidative stress within the cells. As expected, the level of oxidative stress in LNCaP cells increased with Cmp-LAAO treatment as confirmed by 2′, 7′-dichlorofluorescin diacetate (DCFDA) fluorescence assay. Co-treatment with catalase significantly reduced the cytotoxic effect of Cmp-LAAO, thereby affirming that hydrogen peroxide is probably the main mediator of Cmp-LAAO cytotoxicity. Hence, Cmp-LAAO may be a potential cancer therapeutic for prostate cancer.A new L-amino acid oxidase was isolated from the venom of Crotalus mitchellii pyrrhus (Cmp-LAAO).Cmp-LAAO exhibits cytotoxic effect on LNCaP prostate cancer cells.Cmp-LAAO induces apoptosis and oxidative stress in LNCaP cells.