Autophagy, an evolutionarily conserved process, is intricately involved in many aspects of human health and a variety of human diseases, including cancer. Discovery of small-molecule autophagy modulators with potent anticancer effect would be of great significance. To this end, a natural product library consisting of 170 natural compounds were screened as autophagy modulators with potent cytotoxicity in our present study. Among these compounds, gossypol acetate (GAA), the mostly used medicinal form of gossypol, was identified. GAA effectively increased the number of autophagic puncta in GFP-LC3B-labeled 293T cells and significantly decreased cell viability in different cancer cells. In A549 cells, GAA at concentrations below 10 μM triggered caspase-independent cell death via targeting autophagy, as evidenced by elevated LC3 conversion and decreased p62/SQSTM1 levels. Knocking down of LC3 significantly attenuated GAA-induced cell death. Mechanistically, GAA at low concentrations induced autophagy through targeting AMPK-mTORC1-ULK1 signaling. Interestingly, high concentrations of GAA induced LC3 conversion, p62 accumulation, and yellow autophagosome formation, indicating that GAA at high concentrations blocked autophagic flux. Mechanistically, GAA decreased intracellular ATP level and suppressed lysosome activity. Exogenous ATP partially reversed the inhibitory effect of GAA on autophagy, suggesting that decreased ATP level and lysosome activity might be involved in the blocking of autophagy flux by GAA. Collectively, our present study reveals the mechanisms by which GAA modulates autophagy and illustrates whether autophagy regulation by GAA is functionally involved in GAA-induced cancer cell death.