Diazinon (DZ) (O,O-diethyl-O-[2-isopropyl-6-methyl-4-pyrimidinyl]phosphorothioate) is an organophosphate pesticide which is extensively used to control household insects and fruit and vegetable crops. The exposure to this pesticide has been linked to the development of the serious problem in several experimental animals. The contamination of food by DZ may increase its danger to humans. The aim of this study was to investigate the toxic effect of DZ on intestine using an in vitro model (HCT116). Therefore, we evaluated the cell viability, elucidated the generation of free radicals, measured the mitochondrial membrane potential, and valued DNA fragmentation. Our results showed that DZ is cytotoxic to HCT116. It causes oxidative damage through the generation of free radicals and induces lipid peroxidation and DNA fragmentation. We also demonstrated that such effects can be responsible for DZ-induced apoptosis.