Neonicotinoids are the most widely used pesticides in the world. However, research studies have shown that it can affect the cognitive abilities and health of non-target bees and other wild pollinators by inducing DNA damage, apoptosis and mitochondrial damage, injure to its central nervous system, and it is even developmentally neurotoxic to mammals and humans, with mitochondria being an important target of neonicotinoids. Therefore, this article reviews the role of mitochondrial morphology, calcium ions (Ca2+) homeostasis, respiratory function, apoptosis, and DNA damage in neonicotinoids-induced systemic toxicity. Additionally, it evaluates the protective effects of various active substances including vitamin C, N-acetylcysteine (NAC), curcumin (CUR), glutathione reduced (GSH), caffeic acid phenethyl ester (CAPE), resveratrol, and thymoquinone (TQ) on neonicotinoids-induced toxicity. This review manuscript found that mitochondria are important targets to neonicotinoids. Neonicotinoids can cause DNA damage, apoptosis, protein oxidation, and lipid peroxidation in non-target organisms by altering mitochondrial Ca2+ homeostasis, inhibiting mitochondrial respiration, and inducing reactive oxygen species (ROS) production. Several active substances (vitamin C, NAC, CUR, GSH, resveratrol, CAPE, and TQ) play a protective role against neonicotinoid-induced systemic toxicity by inhibiting ROS signaling pathways, apoptosis, and lipid peroxidation. This review manuscript emphasizes the importance and urgency of the development of neonicotinoid antidotes, emphasizes the prospect of the application of targeted mitochondrial antidotes, and prospects the development of neonicotinoid antidotes in order to provide some strategies for the prevention of neonicotinoid toxicity.