Mycobacterium marinum is a major pathogen in aquaculture, posing a substantial threat to the health and sustainability of tongue sole (Cynoglossus semilaevis) farming. This study investigated the genetic basis of immune response in tongue sole by comparing transcriptome profiles of liver and spleen tissues from symptomatic (susceptible) and healthy (resistant) individuals during a natural M. marinum outbreak. Transcriptomic analyses identified differentially expressed genes and enriched pathways related to immune responses. Key genes, including atp6ap1, gpi, and idh3a, were found to be crucial in immune response to M. marinum infection, involved in immune processes such as signal transduction, antigen processing, and metabolic pathways. Protein-protein interaction networks highlighted central hub genes such as nedd8, jun and junb, which play pivotal roles in immune regulation. These findings provide insights into the orchestrated immune responses to mycobacteriosis, which can inform selective breeding strategies for disease-resistant tongue sole strains. This is the first comprehensive transcriptome analysis of M. marinum natural infection in tongue sole, offering valuable data for future research and disease management in aquaculture.