Anoctamin1 (ANO1), a calcium-activated chloride channel (CaCC), has emerged as a promising drug target to alleviate various cancer types, including non-small cell lung cancer (NSCLC) and prostate cancer. ANO1 inhibitors have demonstrated the anticancer effects, but their effects of in synergy with other oncogenic targets have not been fully reported. This study aimed to elucidate the potential of diminishing ANO1 protein levels as a robust anticancer approach. By employing an innovative approach, we screened a novel ANO1downregulator, PIC,a PI3K inhibitor, via a high-throughput yellow fluorescent protein (YFP) reduction assay. The subsequent investigation assessed effects of PICin NSCLC and prostate cancer. Through insilicomolecular docking analyses, we ascertained the superior binding affinity of PICtoward both ANO1 and PI3K, surpassing that of the control ligand. Subsequent immunoblotting experiments validated dose-dependent downregulation of ANO1 protein expression byPICand showcased a reduction in p-AKT levels, indicating effective PI3K pathway inhibition. Differential cell sensitivity became apparent, with ANO1-expressing PC9 and PC3 cells displaying heightened susceptibility to cell proliferation-inhibiting effects exerted by PIC,compared to ANO1-negative Chinese hamster ovary cells and ANO1-knockout PC3cells. Ourfindings emphasize the significance of targeting the ANO1 and PI3K pathways as a novel approach to alleviate NSCLC and prostate cancer.