Aminopeptidase N (APN/CD13) over expressed on tumour cells, plays a critical role in tumour invasion, metastasis and tumour angiogenesis. In this article, we described the design, synthesis and preliminary activity studies of novel 3-amino-2-hydroxyl-3-phenylpropanoic acid derivatives as APN inhibitors. The in vitro enzymatic inhibitions on APN from porcine kidney showed that compound 7e had the most potent inhibitory activity against APN with the IC(50) value to 1.26 ± 0.01 μM, which is better than that of bestatin (IC(50) = 2.55 ± 0.11 μM). In addition, compound 7e also showed better inhibitory activity against APN on human ovary clear cell carcinoma cell ES-2 than bestatin with the IC(50) value to 30.19 ± 1.02 μM versus 60.61 ± 0.1 μM. Compound 7e could be used as the lead compound in the future for anti-cancer agent research.