Abstract: Infectious hematopoietic necrosis virus (IHNV) is an important pathogen that seriously threatens the rainbow trout aquaculture industry. Astragalus polysaccharides (APS), as natural immunomodulatory adjuvant, can enhance host immune responses. This study aimed to evaluate the regulatory effect of APS combined with an inactivated IHNV vaccine on immune responses in the skin-associated lymphoid tissue (SALT) of rainbow trout. The skin mucosal immune response was analyzed via vaccination and challenge experiments, combined with histopathological observation, transcriptome sequencing, and quantitative real-time PCR. Histopathological observation showed that APS combined with the inactivated vaccine alleviated IHNV-induced skin tissue damage. Transcriptome analysis revealed that, compared with the vaccine-only group, differentially expressed genes in the skin tissue of the APS-adjuvanted vaccine group at the early immunization stage (D7) were mainly enriched in metabolism-related pathways, including pyrimidine metabolism and purine metabolism. At the post-challenge stage (D21), they were mainly enriched in the pentose phosphate pathway, arginine and proline metabolism, and calcium signaling pathway. Expression analysis of key gene showed that, compared with the vaccine-only group, antiviral-related genes such as IFN-1a and Mx1 showed earlier expression responses in the APS-adjuvanted vaccine group during the immunization stage; from D21 after challenge onward, immune-related genes such as IgM, IgT, and IFN-γ exhibited relatively higher expression levels. In conclusion, these findings indicate that APS enhances the skin mucosal immune response induced by the inactivated IHNV vaccine in rainbow trout.