We report in this study the identification of a natural product-like antagonist (la) of Vps34 as a potent autophagy modulator via structure-based virtual screening. Aurone derivative la strongly inhibited Vps34 activity in cell-free and cell-based assays. Significantly, la prevents autophagy in human cells induced either by starvation or by an mTOR inhibitor. In silico modeling and kinetic data revealed that la could function as an ATP-competitive inhibitor of Vps34. Moreover, it suppressed autophagy in vivo and without inducing heart or liver damage in mice. la could be utilized as a new motif for more selective and efficacious antagonists of Vps34 for the potential treatment of autophagy-related human diseases.