Parasitic infections, such as those caused by the myxozoans and , pose major threats to wild and farmed salmonids due to severe tissue damage and impairment of the host immune system. While individual infections have been studied, limited information is available on the host response during co-infection. This study investigated the transcriptomic immune response of rainbow trout () during single and sequential co-infections with and using RNA-seq. Trout were exposed to single infections (Mc or Tb) followed by co-infections (Mc or Tb). Fish were sampled at 31 days post-single infection (1 day post-co-infection). RNA from gill and caudal fin (portal of parasite entry) was sequenced, followed by differentially expressed genes (DEGs) identification and GO and KEGG enrichment. In the caudal fin, Mc (1 day after co-infection with ) fish showed mild immune activation with upregulation, while Tb fish exhibited a stronger response involving signaling. In gills, Mc fish showed moderate cytokine-related gene upregulation, while Tb (1 day after co-infection with ) fish displayed increased expression of humoral response genes (, immunoglobulin pathways) but suppression of genes involved in B cell development. These results indicate that the order of infection shapes the outcome of the host immune response, offering candidate targets at the host-pathogen interface.