[Objectives] Aminopeptidase N (APN) is an important receptor ofBacillus thuringiensis(Bt) in the midgut of insects that is associated with Bt toxicity and the resistance mechanisms of insect to Bt protein. Cloning and spatio-temporal expression of aminopeptidase N encoding genes inCnaphalocrocis medinalis(Guenée) (Lepidoptera: Pyralidae) were conducted to reveal the function ofCsAPNsand the molecular resistance mechanism ofC. medinalisto Bt protein.[Methods] The full-length cDNA sequence encoding APN was cloned by degenerative primer PCR and RACE techniques. The relative expression levels ofAPNgenes in different developmental stages and various larval tissues ofC. medinaliswere determined by Quantitative Real-time PCR method.[Results] Comparison of sequence identity in NCBI revealed fourAPNgenes belonging to different classes of the APN family were namedCmAPN1(GenBank Accession No. HQ853294),CmAPN2(GenBank Accession No. HQ853295),CmAPN3(GenBank Accession No. KJ143755) andCmAPN4(GenBank Accession No. HQ853296), respectively. The full-length sequences encodingCmAPN1-4were 3 698, 3 478, 3 150 and 3 149 bp, respectively. The open reading frames were 3 045, 2 877, 3 045 and 2 862 bp, which encoded proteins of 965, 958, 1 014 and 952 amino acid residues, respectively. Putative protein sequences included potential glycosylation sites and a zinc metal binding site motif of HEX2HX18E, which is typical of the active sites of the zinc-dependent metalloproteases, and a highly conserved GAMEN motif, which was to form part of the active site. Each sequences had a cleavable N-terminal signal peptide and glycosylphosphatidylinositol (GPI) anchor signal peptide, which is also typical characters of lepidopteran APN proteins. Expression levels ofCmAPNsin larval stages were significantly higher than these in eggs, pupae or adults. During the larval stage, the expression level ofCmAPN1was significantly lower than that ofCmAPN2-4, though it differed significantly among different larval developmental stages. Transcription levels ofCmAPN2-4increased with larval instars.CmAPNspresented significantly different expression levels in different larval tissues, with higher expression in the gut and abdomen and lower expression in the head, thorax, fat body, and entire body minus gut tissues.CmAPN1andCmAPN2showed higher expression in the midgut and hindgut, respectively. Similarly,CmAPN3exhibited significantly high transcription levels in both the foregut and midgut. However, significantly lower expression levels ofCmAPN4were detected among different tissues compared to those ofCmAPN1-3.[Conclusion] CmAPNshave significantly differently spatio-temporal expression patterns in different developmental stages and tissues ofC. medinalis. Therefore, to determine the function ofCmAPNsit is very important to choose the appropriate instar for RNA interference.