Aminopeptidase N (APN) is important in tumour processes. The present study detected the anti‑tumour activity of the novel APN inhibitor DH‑12a, which is an indoline‑2,3‑dione derivative. In the present study, Bestatin, a clinical APN inhibitor was used as a positive control. The expression of APN in the ES-2 and 3AO cell lines were assessed using flow cytometry and the drug inhibition constants of DH‑12a (Ki=13.15 µM) and Bestatin (Ki=16.57 µM) were assessed using a double reciprocal method of competitive inhibition. The in vitro effects of DH‑12a on cell proliferation were assessed using a 3‑(4,5‑dimethyl‑thiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide assay on human cell lines of ES‑2 (IC50=43.8 µM), A549 (inhibition rate=41.5% at 160 µM DH‑12a), HL60 (inhibition rate=47.83% at 160 µM DH‑12a) and 3AO (IC50=70.2 µM). The inhibition rates were consistently higher than those of Bestatin. The effects of DH‑12a on cell migration (inhibition rates in ES‑2 cells and 3AO cells were 56.4 and 76.5%, respectively at 15 µM) and invasion (inhibition rates in ES‑2 cells and 3AO cells were 75.6 and 66.5%, respectively at 15 µM) were assessed using transwell plates. The in vivo effects of DH‑12a on tumour proliferation and lung tumour metastasis were determined using an H22 xenograft mice model, where DH‑12a was administered in combination with genotoxic 5‑fluorouracil. The anti‑tumour activities of DH‑12a in vivo were also greater than those of Bestatin. In conclusion, the in vitro effects of DH‑12a on tumour proliferation, migration and invasion were consistent with the in vivo effects. In addition, DH‑12a exhibited greater anti‑tumour properties compared with Bestatin.