Aminopeptidase N(APN) is a member of the zinc metalloproteinase M1 family. APNs, located on the cell membrane of brush border membrane vesicles(BBMV) in Lepidoptera insects midgut, not only play an important role in the process of protein digestion and absorption, but also is an important receptor protein for Bt. In this study, seven APN genes with the full sequence of 2592-3099 bp from Helicoverpa armigera(Ha APN1-7, Genbank acc. No. MT002819-MT002825) were cloned by PCR. Bioinformatics analysis results showed that HaAPN1-7 encoded 863-1032 amino acid with a molecular weight of 110-115 kDa and an isoelectric point of 4.7-6.4, and signal peptides of 15-20 aa at the N-terminal. Phylogenetic analysis showed that HaAPN1-7 clustered into Class 1-7 families. RT-qPCR results showed that the expression level of APN2 was the highest and that of APN7 was the lowest in midgut of the fifth instar larvae from the susceptible strain. Similar APN expression pattern was also found in Cry1 Ac-resistant strain. The APN activity in midgut juice and BBMV both significantly decreased when the larvae were fed Cry1 Ac and Vip3 Aa protein. But when the larvae were fed Cry2 Ab protein, the APN activity was clearly reduced only in midgut BBMV. The APN activity in midgut juice and BBMV also significantly changed after H. armigera had tolerance to Cry1 Ac, Cry2 Ab, or Vip3 Aa. It is suggested the different types of HaAPNs may play different roles in the insecticidal mechanism of Bt, and the change of APN activity may be related to Bt degradation and resistance evolution.