The fall armyworm, Spodoptera frugiperda, is an invasive species of significant concern due to its highly destructive nature and the substantial threat it poses to food crops. Bats are one of the important natural enemies of lepidopteran insects, controlling their survival and quantity. To investigate whether and how S. frugiperda responds to the ultrasound emitted by insectivorous bats in newly invaded areas after arriving at a new invasion area, this study first observed and recorded the behavioral changes of S. frugiperda both before and after exposure to ultrasound emitted by local bats. Then, the transcriptomes of one-day-old male adults of S. frugiperda being exposed to bat ultrasound for 0.5, 3, 12, 24, 48 and 72 hours were compared and analyzed with those in normal environment as controls. The comprehensive analysis of the transcriptomes provided valuable information into the molecular changes that occur in S. frugiperda in response to bat ultrasonic signals. The behavioral results indicated that local bats ultrasound which were within the hearing range of S. frugiperda could significantly reduce the behavioral activity of moths, suggesting that these signals may play a crucial role in the moths behavior. The transcriptome analysis showed that bats ultrasonic stress induced extensive changes in gene expression in the body of S. frugiperda. Compared with the control group, a total of 2389 significantly up-regulated genes and 2491 significantly down-regulated genes were identified under different durations of ultrasound stimulation. The 521 genes that were significantly up-regulated or down-regulated at multiple time points were defined as key genes in response to bat ultrasound. GO and KEGG enrichment analyses of these key genes further revealed the different processes affected by bat ultrasonic stress in S. frugiperda. The results showed that 215 up-regulated genes were highly correlated with processes such as neural signal transmission, sound perception, auditory receptor cell development, oxidative stress and fear response, while 306 down-regulated genes were mainly enriched in various energy metabolism pathways and biosynthesis processes. These findings suggest that bat ultrasonic signals may have a profound impact on the physiology and behavior of S. frugiperda. To further validate the reliability of the transcriptome analysis results, qRT-PCR experiments were conducted. The study provides new insights into the biological and molecular mechanisms of S. frugiperda in response to bat ultrasonic stress, and could provide greater support for developing more effective and environmentally friendly methods for controlling S. frugiperda and other lepidopteran pest species in the future.