We recently developed a specific small molecule inhibitor of aminopeptidase N (APN), named as HNSA, through a high throughput screening. In the present study, we investigated the major cellular phenotypes of HNSA in comparison with those of APN knock-down in human fibrosarcoma cells and the mechanism of angiogenesis inhibition by the compound using DNA microarray analyses. Global gene expression analyses showed that HNSA signatures are significantly correlated with those of APN knock-down in HT1080 cells, suggesting that APN is a primary target of HNSA in the cells. Using the angiogenesis-focused DNA microarrays, nine of angiogenesis-related genes were identified as crucial mediators of angiogenesis inhibition by HNSA. These data demonstrate that HNSA can be used as a valuable tool to decipher the APN function in angiogenesis. © 2008 Elsevier Inc. All rights reserved.