The calcium-activated chloride channel ANO1, also known as TMEM16A, shows calcium-dependent activation. The channel is expressed broadly and contributes to a variety of physiological processes. The mutation or abnormal expression of ANO1 channel is related to cancer and gastrointestinal dysfunction. Therefore, this channel is promising as a drug target for the above-mentioned diseases. Revealing the allosteric mechanism and the drug regulation mechanism of ANO1 is very important for understanding the relationship between channel structure and function. First, we explored the Ca²⁺-dependent gating mechanism of the ANO1 channel by molecular simulations. The results show that chloride ions remain semi-hydrated as they pass through the hydrophobic neck region and require the assistance of K645 and K588. Moreover, E705 in the TM7 plays a key role in Ca²⁺ dependent activation. It stabilizes the closed conformation of the pore in the Ca²⁺ unbound state, but swings 100^o to serve as Ca²⁺ binding coordination in the Ca²⁺ bound state. Secondly, we identified the binding pocket of the ANO1 inhibitor, CaCC_inh-A01, which is located at the extracellular entrance of the pore by molecular docking and targeted mutagenesis. To characterize the druggability of this binding pocket, we performed a virtual screen and found a highly potent inhibitor of ANO1, theaflavin. Molecular dynamics simulations revealed that theaflavin adopts a "wedge insertion mode" to block the ion conduction pore and induces pore closure. Moreover, the binding mode showed that the theaflavin pedestal plays an important role in pore blockade, and R515, R535, T539, K603, E623, and E633 were determined to be most likely to interact directly with the pedestal. Next, to confirmed that ANO1 can be used as an anticancer drug target, we obtained a natural product inhibitor of ANO1, arctigenin, based on the inhibitor binding pocket identified in the above study. Molecular biology experiments showed that arctigenin concentration-dependently inhibited the proliferation and migration of LA795, however, the inhibition effect can be abolished by knockdown of the endogenous ANO1 with shRNA. Further, we injected arctigenin on xenograft mouse model which exhibited significant antitumor activity with no adverse effect. Finally, to confirmed that ANO1 can be a drug target for the treatment of gastrointestinal motility disorders, we screened for several ginsenoside analogs (GRb1 GRg2 and GRf) that activate ANO1 channels. Isolated guinea pig ileum assay showed both GRb1 GRg2 and GRf increased the amplitude and frequency of ileum contractions. Therefore, GRb1 GRg2 and GRf can be considered a lead compound for the development of novel drugs for the treatment of diseases caused by ANO1 dysfunction. In summary, based on the study of a series of gating and drug regulatory mechanisms of ANO1 channels, we confirmed the feasibility of ANO1 as a drug target for diseases such as cancer and gastrointestinal dyskinesia, which will be useful for drug development of related diseases.