Macroautophagy is a cellular process whereby the cell sequesters and recycles cytosolic constituents in a lysosome-dependent manner. It has also been implicated in a number of disorders, including cancer and neurodegeneration. Although a previous report that AGS3 over-expression promotes macroautophagy suggests a stimulatory role of AGS3 in this process, we have found that knock-down of AGS3, unexpectedly, also induces macroautophagy, indicating an inhibitory function of endogenous AGS3 in macroautophagy. Interestingly, AGS3 phosphorylation is decreased upon induction of mammalian target of rapamycin (mTOR)-dependent macroautophagy. Moreover, unlike wild-type AGS3, over-expression of an AGS3 mutant lacking this modification fails to enhance macroautophagic activity. These observations imply that AGS3 phosphorylation may participate in the modulation of macroautophagy.