To better understand the molecular mechanisms underlying allergens and parasite immunity and discover the stage-enriched gene expression of fish-borne zoonotic nematodes in the stomach, we used RNA-seq to study the transcriptome profiles of Anisakis pegreffii (Nematoda: Anisakidae, AP) in simulated gastric juice. Mobile L3 larvae were incubated in simulated medium at 37 °C in 5% CO2 (AP-GJ) and the control group larvae were collected in PBS under the same conditions (AP-PBS). We found that the sequences of A. pegreffii were highly similar to Toxocara canis sequences. Among the transcripts, there would be 138 up-regulated putative genes and 251 down-regulated putative genes in AP-GJ group. Several lipid binging-related genes were more highly expressed in AP-GJ larvae. Moreover, 17 allergen genes were up-regulated and 29 were down-regulated in AP-GJ larvae. Eleven allergen genes belonged to one or more of the following three categories: biological process, cellular component, and molecular function. According to KEGG analysis, the main pathways that were represented included protein processing in transcription, immune system, cancer, and infectious disease. In particular, the most significant changes in the expression of parasite-derived allergen products occurred in AP-GJ larvae. This study helps us to extend our understanding of the biology of the fish-borne zoonotic parasite A. pegreffii and could be helpful for more precise risk assessment and providing guidelines for allergic consumers. Copyright © 2020 Elsevier B.V. All rights reserved.