Abstract  [Aim]  To assess the effect of RNA interference of the siRNA delivery strategy on Mythimna separata using rolling circle transcription. [Methods]  We used qPCR to determine the silencing efficiency of the two target genes, Msα-tubulin, and Msβ-tubulin. The RNAi Feeding Bioassay was used to observe changes in M. separata body weight, body length, and lethal phenotypes. Additionally, the 3rd instar larvae were treated with RNA microspheres, which were synthesized using the rolling circle transcription method. [Results]  After 24 h of RNA microsphere treatment, Msα-tubulin and Msβ-tubulin expression decreased by 51.6% and 44.3%, respectively, demonstrating the effectiveness of the target gene silencing. After 48 h there was a further 32% decrease in the expression of Msα-tubulin, which then increased by 134.4% at 72 h. After 48 and 72 h of treatment, the expression level of Msβ-tubulin was upregulated by 120.5% and 129.0%, respectively. The RNAi feeding bioassay results showed that after 7 days, larval body weight and body length decreased by 51.1% and 34.9%, respectively in the Msα-tubulin treatment group, and decreased by 49.0% and 32.4% in the Msβ-tubulin treatment group, respectively, compared to the control. After 14 days, the mortality rate of larvae reached 42.2% for both the Msα-tubulin and Msβ-tubulin treatment groups. [Conclusion]  RNA microspheres, created via the rolling circle transcription method, can trigger an RNAi response in M. separata, interfere with the expression of target genes, significantly increase M. separata mortality, and reduce larval weight and body length. The siRNA delivery strategy, utilizing rolling circle transcription, offers a novel approach to controlling M. separata.