Sulfonylureas are widely used oral anti-diabetic drugs. However, its long-term usage effects on patients' lifespan remain controversial, with no reports of influence on animal longevity. Hence, the anti-aging effects of chlorpropamide along with glimepiride, glibenclamide, and tolbutamide were studied with special emphasis on the interaction of chlorpropamide with mitochondrial ATP-sensitive K⁺ (mitoK-ATP) channels and mitochondrial complex II. Chlorpropamide delayed aging in Caenorhabditis elegans, human lung fibroblast MRC-5 cells and reduced doxorubicin-induced senescence in both MRC-5 cells and mice. In addition, the mitochondrial membrane potential and ATP levels were significantly increased in chlorpropamide-treated worms, which is consistent with the function of its reported targets, mitoK-ATP channels. Increased levels of mitochondrial reactive oxygen species (mtROS) were observed in chlorpropamide-treated worms. Moreover, the lifespan extension by chlorpropamide required complex II and increased mtROS levels, indicating that chlorpropamide acts on complex II directly or indirectly via mitoK-ATP to increase the production of mtROS as a pro-longevity signal. This study provides mechanistic insight into the anti-aging effects of sulfonylureas in C. elegans.