Foodborne pathogens are the leading causes of human diseases that result in hundreds of thousands of deaths annually. Clostridium perfringens is a notorious foodborne pathogen of global significance that is known to produce a large repertoire of toxic factors, several of which can severely destroy the gut lining to lead to abdominal pain, diarrhea, and even death. However, like many other foodborne pathogens, there is no specific treatment for infections caused by C. perfringens and rehydration therapy is largely ineffective at reducing symptoms and preventing transmission. Given this, there is an urgent need to discover new therapeutic targets that can be harnessed to treat C. perfringens infection. We recently discovered that a highly toxic factor produced by C. perfringens named perfringolysin O (PFO) activates the innate immune sensor NLR family pyrin domain containing 3 (NLRP3). We characterized that PFO is the specific agonist of NLRP3 inflammasome and mechanistically, the intracellular entrance of PFO is required for active assembly of NLRP3 inflammasome. Further, PFO targets the mitochondria resulting in mitochondrial destabilization and reactive oxygen species (ROS) release. Mito-TEMPO scavenging mitochondrial ROS (mtROS) specifically inhibits the level of Caspase-1 p20 and interleukin (IL)-1β in both in vitro and in vivo models. Additionally, 5-hydroxydecanoate (5-HD), a selective mitochondrial ATP-sensitive potassium channel (KATP) inhibitor, could relieve PFO-induced NLRP3 inflammasome activation as well as mitochondrial destabilization, indicating that potassium ion flux is necessary for mitochondrial damage upstream of NLRP3 inflammasome activation. We expect that the information gathered from this study will develop insights into features of the immune response that can be targeted for the treatment of food poisoning and other foodborne pathogenic infections.