Abstract: Autophagy is a lysosomal degradation pathway, and plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. Here, combined with allosteric site identification methods, high-throughput virtual screening, and in vitro activity evaluation, we found that compound 10 can activate autophagy and has good anti-MDA-MB-231 cell proliferation activity (the half maximal inhibitory concentration IC₅₀=8.25±1.53 μmol·L^-1). Subsequently, molecular docking, molecular dynamics simulation, and immunoblotting assay demonstrate that compound 10 can target and activate beclin-1. In vitro studies have shown that compound 10 can induce autophagy-associated cell death in MDA-MB-231 cells. In addition, it was found that compound 10 can induce apoptosis in MDA-MB-231 cells. Taken together, we identified the candidate compound 10 as an effective and selective targeting beclin-1 to activate autophagy as a lead compound, which provide a reference for further development and optimization of small molecule drugs targeting beclin-1 to activate autophagy for clinical treatment.