Aminopeptidase N(APN) has been shown as a receptor of several coronaviruses, such as HCoV-229E, TGEV, CCoV and FeCoV. Bestatin and Actinonin are inhibitors which can block APN enzymatic activity. These inhibitors bind to the catalytic site of APN, while viruses bind to the outer surface of APN. Here we investigate the mechanism of APN inhibition on protein-protein binding, receptor expression and coronavirus entry. We find that these chemical compounds can inhibit the protein-protein interaction between APN and Coronavirus spike; these inhibitors can also regulate APN RNA and protein expression; additionally, these compounds can inhibit the pseudovirus entry of HCoV-229E into human cells at a certain level. Additionally, coronavirus spike-treated human cells show a decrease in APN expression. This phenomenon may reveal an adaptation of cells to the different treatments and conditions. Our research may provide a new potential strategy for antiviral treatment.