Protein synthesis inhibitors such as cycloheximide (CHX) are known to suppress protein degradation including autophagy. The fact that CHX inhibits autophagy has been generally interpreted to indicate that newly synthesized protein is indispensable for autophagy. However, CHX is also known to increase the intracellular level of amino acids and activate mTORC1 activity, a master negative regulator of autophagy. Accordingly, CHX can affect autophagic activity through inhibition of de novo protein synthesis and/or modulation of mTORC1 signaling. In this study, we investigated the effects of CHX on autophagy using specific autophagy markers. We found that CHX inhibited starvation-induced autophagy but not Torin1-induced autophagy. CHX also suppressed starvation-induced puncta formation of GFP-ULK1, an early-step marker of the autophagic process which is regulated by mTORC1. CHX activated mTORC1 even under autophagy-inducible starvation conditions. Finally, the inhibitory effect of CHX on starvation-induced autophagy was cancelled by the mTOR inhibitor Torin1. These results suggest that CHX inhibits starvation-induced autophagy through mTORC1 activation and also that autophagy does not require new protein synthesis at least in the acute phase of starvation. (C) 2014 Elsevier Inc. All rights reserved.