[Aim]  To identify the AiJHAMT2 gene in Agrotis ipsilon, analyze its sequence characteristics and clarify of its biological functions. [Methods]  The cDNA sequence of AiJHAMT2 was identified from the A. ipsilon transcriptome using a homology search method. The sequence characteristics of AiJHAMT2 were analyzed using bioinformatic software. AiJHAMT2 was silenced with RNA interference technology and subsequent changes in juvenile hormone (JH) titer, larval survival rate, body length, body weight, and pupation, and emergence rates, were assessed. [Results]  AiJHAMT2 contained an open reading frame of 855 bp. The protein encoded by AiJHAMT2 possessed features typical of other insect JHAMTs, including a S-adenosyl methionine-binding motif and 11 conserved substrate-interacting residues. The AiJHAMT2 gene was localized on chromosome 12, and its genomic sequence spans 5 673 bp, comprising four exons and three introns. Phylogenetic analysis revealed that AiJHAMT2 is most closely related to Mythimna separata MsJHAMT2. Injection with double-stranded RNA (dsRNA) targeting AiJHAMT2 effectively silenced the gene in 3rd instar larvae. Expression of AiJHAMT2 significantly decreased by 74.0% (P=0.026) and 76.6% (P=0.008), respectively, 24 and 48 h post-injection compared to the control group, whereas expression of AiJHAMT1 significantly increased by 2.21-fold compared to the control group 48 h post-injection (P=0.009). Silencing AiJHAMT2 significant reduced the JH titer of larvae by 46.4% (P=0.020). This also significantly reduced larval survival rate, body length, body weight, and pupation rate by 28.1% (P<0.001), 31.7% (P=0.007), 36.3% (P=0.002), and 35.2% (P<0.001), respectively. However, silencing AiJHAMT2 had no significant effect on the emergence rate (P=0.252). [Conclusion]  Silencing AiJHAMT2 significantly reduced larval survival rate and impaired larval growth and pupation, demonstrating that this gene is a potential RNAi target for controlling A. ipsilon.