Down syndrome (DS) is linked with mitochondrial dysfunction, which results in accumulation of damaged mitochondria. This study revealed that mitophagy, a form of selective autophagy activated to clear damaged mitochondria, is deficient in primary human fibroblasts derived from individuals with DS leading to accumulation of damaged mitochondria with consequent increases in oxidative stress. RNA sequencing was performed on nine 2N and eight DS human fibroblasts samples of age 5 months and 2 years. These samples came from five unrelated 2N individuals and three unrelated DS individuals. The study found that mammalian target of rapamycin is strongly hyperactivated, which suppresses macroautophagy induction and the transcriptional expression of proteins important for autophagosome formation.