[Objectives]  To investigate the toxicity of lufenuron and its effects on the growth and development of Spodoptera frugiperda larvae. [Methods]  A bioassay was conducted to investigate the toxicity of lufenuron to S. frugiperda larvae and the chitin content and chitinase activity of larvae exposed to sublethal concentrations of lufenuron were measured using the glucosamine, and N-acetylglucosamine methods, respectively. Bioinformatics was used to screen chitinase genes from the transcriptome database for phylogenetic analysis, after which real-time fluorescence quantitative PCR was used to analyze the effect of lufenuron on the expression of these genes. [Results]  Larvae gradually became less active and molting and cuticle development became abnormal. Larvae eventually became deformed and died. The lethal 20% (LC₂₀) and 50% (LC₅₀) concentrations of lufenuron after 96 h were 0.517 mg/L and 1.009 mg/L, respectively. Chitin content and chitinase activity were significantly reduced after 48 h and 96 h exposure to sublethal concentrations of lufenuron. A phylogenetic tree indicates that the five chitinase genes in the NR database are different types. qPCR analysis shows that the LC₂₀ concentration of lufenuron can activate chitinase gene expression, whereas the LC₅₀ concentration can inhibit it. [Conclusion]  Lufenuron reduces chitin content, inhibits chitinase activity and disrupts chitinase gene expression, in S. frugiperda larvae. It therefore has potential as an insecticide for the control of this important global pest.