Clothianidin (CLO) is a high-frequently detected neonicotinoid pesticide in fruits and vegetables, whose exposure security deserves attention. This study evaluated the apoptotic toxicity of CLO on Caco-2 cells at doses of 100 nM, 10 μM, and 1 mM. After exposure, CLO induced to a remarkable change of signaling proteins that participated in the process of cell apoptosis, including caspase 3, cleaved-caspase 3, and caspase 9. CLO treatment further induced a decrease of mitochondrial membrane potential and increased the protein level of cytochrome C. Reactive oxygen species (ROS) and intracellular Ca