This study demonstrates the role of the NAD-dependent deacetylase Sirt1 in regulating autophagy. Transient overexpression of wild-type Sirt1 is sufficient to stimulate basal rates of autophagy, while Sirt1−/− mouse embryonic fibroblasts fail to fully activate autophagy under starved conditions. Sirt1 forms a molecular complex with essential components of the autophagy machinery, including Atg5, Atg7, and Atg8, and can directly deacetylate these components in an NAD-dependent manner. The absence of Sirt1 leads to elevated acetylation of proteins required for autophagy in both cultured cells and embryonic and neonatal tissues. Sirt1−/− mice exhibit phenotypes similar to Atg5−/− mice, including the accumulation of damaged organelles, disruption of energy homeostasis, and early perinatal mortality. In utero delivery of the metabolic substrate pyruvate extends the survival of Sirt1−/− pups. These findings suggest that Sirt1 is an important regulator of autophagy in vivo and provide a link between sirtuin function and the cellular response to limited nutrients.This study demonstrates the role of the NAD-dependent deacetylase Sirt1 in regulating autophagy. Transient overexpression of wild-type Sirt1 is sufficient to stimulate basal rates of autophagy, while Sirt1−/− mouse embryonic fibroblasts fail to fully activate autophagy under starved conditions. Sirt1 forms a molecular complex with essential components of the autophagy machinery, including Atg5, Atg7, and Atg8, and can directly deacetylate these components in an NAD-dependent manner. The absence of Sirt1 leads to elevated acetylation of proteins required for autophagy in both cultured cells and embryonic and neonatal tissues. Sirt1−/− mice exhibit phenotypes similar to Atg5−/− mice, including the accumulation of damaged organelles, disruption of energy homeostasis, and early perinatal mortality. In utero delivery of the metabolic substrate pyruvate extends the survival of Sirt1−/− pups. These findings suggest that Sirt1 is an important regulator of autophagy in vivo and provide a link between sirtuin function and the cellular response to limited nutrients.