The study investigates the role of mitochondrial DNA (mtDNA) mutations in intestinal aging in mice. It finds that increased mtDNA mutation burden in the small intestine of aged male mice leads to a decline in intestinal homeostasis, characterized by decreased intestinal crypt number, increased villus length, higher expression of CDKN1A/p21, and shorter telomere length. This is associated with impaired Wnt/β-catenin signaling, reduced LGR5-expressing intestinal stem cells (ISCs), and decreased organoid formation efficiency. Mechanistically, the increased mtDNA mutation burden triggers NAD+ depletion, which activates the ATF5-dependent mitochondrial unfolded protein response (UPRmt). This UPRmt activation impairs Wnt/β-catenin signaling, leading to ISC exhaustion. Replenishing NAD+ with nicotinamide mononucleotide (NMN) alleviates these aging phenotypes, suggesting a potential therapeutic target for intestinal aging. The study also reveals that the ISR, regulated by NAD+ depletion, activates UPRmt, providing a mechanism linking mtDNA mutation burden to intestinal aging.The study investigates the role of mitochondrial DNA (mtDNA) mutations in intestinal aging in mice. It finds that increased mtDNA mutation burden in the small intestine of aged male mice leads to a decline in intestinal homeostasis, characterized by decreased intestinal crypt number, increased villus length, higher expression of CDKN1A/p21, and shorter telomere length. This is associated with impaired Wnt/β-catenin signaling, reduced LGR5-expressing intestinal stem cells (ISCs), and decreased organoid formation efficiency. Mechanistically, the increased mtDNA mutation burden triggers NAD+ depletion, which activates the ATF5-dependent mitochondrial unfolded protein response (UPRmt). This UPRmt activation impairs Wnt/β-catenin signaling, leading to ISC exhaustion. Replenishing NAD+ with nicotinamide mononucleotide (NMN) alleviates these aging phenotypes, suggesting a potential therapeutic target for intestinal aging. The study also reveals that the ISR, regulated by NAD+ depletion, activates UPRmt, providing a mechanism linking mtDNA mutation burden to intestinal aging.