Aminopeptidase N (APN) is a transmembrane metallopeptidase, which participates in the tumor progress such as proliferation, attachment, angiogenesis and tumor invasion. All of this makes APN as a good chemical therapeutic anti-tumor target. In the present study, we got a novel compound 16l which markedly inhibited the enzyme activity of porcine APN, and the inhibition constant, K(i), of 16l are similar to the positive medicine Bestatin determined using porcine APN. However, when tested using human tumor cells, 16l couldn't effectively inhibit the enzyme activity, cell viability, cell migration and invasion. Computer aided drug design verified that because of the difference in structure, the binding pattern of 16l in the active site of homo sapien and porcine APN was different. The compound 16l could effectively inhibit the enzyme activity of porcine APN, but not homo sapien APN located on the surface of tumor cells. Therefore, the activity screening of APN inhibitor using aminopeptidase N from porcine should be only preliminary determination. The real activity screening should be determined using homo sapien aminopeptidase N.