For many years, ionophores have been used to control coccidiosis in poultry. However, misuse of ionophores can cause toxicity with significant clinical symptoms. The most critical factors influencing ionophores’ toxicity are administration dose, species, and animal age. Although clinical signs of ionophore intoxication are well studied, the toxicity mechanisms of the ionophores at the molecular level still are not fully elucidated. This review summarizes the studies focused on polyether ionophores toxicity mechanisms in animals at the clinical and molecular levels. Studies show that ionophore toxicity mainly affects myocardial and skeletal muscle cells. The molecular mechanism of the toxication could be explained by the inhibition of oxidative phosphorylation via dysregulation of ion concentration. Tiamulin-ionophore interaction and the synergetic effect of tiamulin in ionophore biotransformation are discussed. Furthermore, in recent years ionophores were candidates for reprofiling as antibacterial and anti-cancer drugs. Identifying ionophores’ toxicity mechanisms at the cellular level will likely help develop novel therapies in veterinary and human medicine.