Autophagy is an evolutionarily conserved catabolic process that involves the invagination and degradation of cytoplasmic components through an autophagosomelysosome track. Autophagy functions as a quality control of cellular milieu and is implicated in a wide variety of pathological conditions. However, excessive or imbalanced autophagic flux may also be associated with cellular toxicity and may potentially contribute to the development of pathological conditions. Just as all membrane trafficking systems need to constantly strike a balance in their level of activation and inhibition to ensure proper spatial and temporal delivery of their cargo, autophagy must also be tightly regulated. Here, we provide an overview of the current knowledge regarding the negative regulation of mammalian autophagy in an effort to understand its physiological relevance and potential clinical importance.