Aging in mammals is associated with intestinal homeostasis imbalance and accumulation of mitochondrial DNA (mtDNA) mutations. This study shows that mtDNA mutations in the small intestine of aged mice are linked to physiological intestinal aging. Using POLG mutator mice, researchers found that increased mtDNA mutation burden triggers an ATF5-dependent mitochondrial unfolded protein response (UPRmt), leading to intestinal aging. NAD+ supplementation reversed this aging phenotype, indicating that NAD+ depletion is a key mediator of mtDNA mutation-induced intestinal aging. The study reveals that mtDNA mutations impair intestinal stem cell (ISC) function by reducing colony formation efficiency and increasing apoptosis. This is linked to NAD+ depletion, which activates the integrated stress response (ISR), impairing Wnt/β-catenin signaling and reducing ISC regeneration. The ISR also activates UPRmt through ATF5, leading to mitochondrial dysfunction and further aging. The findings suggest that mtDNA mutations cause intestinal aging by depleting NAD+, activating ISR, and impairing Wnt/β-catenin signaling. NAD+ repletion can reverse these effects, highlighting the role of NAD+ in maintaining intestinal homeostasis. The study provides a mechanism linking mtDNA mutations to intestinal aging and supports NAD+ as a potential biomarker for monitoring tissue aging.Aging in mammals is associated with intestinal homeostasis imbalance and accumulation of mitochondrial DNA (mtDNA) mutations. This study shows that mtDNA mutations in the small intestine of aged mice are linked to physiological intestinal aging. Using POLG mutator mice, researchers found that increased mtDNA mutation burden triggers an ATF5-dependent mitochondrial unfolded protein response (UPRmt), leading to intestinal aging. NAD+ supplementation reversed this aging phenotype, indicating that NAD+ depletion is a key mediator of mtDNA mutation-induced intestinal aging. The study reveals that mtDNA mutations impair intestinal stem cell (ISC) function by reducing colony formation efficiency and increasing apoptosis. This is linked to NAD+ depletion, which activates the integrated stress response (ISR), impairing Wnt/β-catenin signaling and reducing ISC regeneration. The ISR also activates UPRmt through ATF5, leading to mitochondrial dysfunction and further aging. The findings suggest that mtDNA mutations cause intestinal aging by depleting NAD+, activating ISR, and impairing Wnt/β-catenin signaling. NAD+ repletion can reverse these effects, highlighting the role of NAD+ in maintaining intestinal homeostasis. The study provides a mechanism linking mtDNA mutations to intestinal aging and supports NAD+ as a potential biomarker for monitoring tissue aging.