AbstractFlavobacterium covae (columnaris) is the most detrimental bacterial disease affecting the largemouth bass (Micropterus salmoides Lacépède) aquaculture industry. In the current study, fish received an intraperitoneal injection of either 1× PBS (100 μL), LPS in PBS (100 μL, 10 μg/mL), or F. covae (100 μL, 2.85 × 1011 CFU/mL) to simulate immunological challenges. After 24 h post‐injection, liver tissue from the control and treated groups were then collected for transcriptome analysis. Results of the Gene Ontology (GO) and KEGG pathway analyses for the F. covae and LPS‐injected groups found differentially expressed genes (DEGs) enriched primarily in toll‐like receptors (TLRs), cytokine–cytokine receptors, complement and coagulation cascades, and the PPAR signalling pathways. This suggests that the liver immune system is enhanced by these five combined pathways. Additionally, the DEGs TLR5, MYD88, and IL‐1 were significantly upregulated in F. covae and LPS‐injected fish compared to the controls, whereas IL‐8 was downregulated. The upregulation of TLR5 was unexpected as F. covae lacks flagellin, the protein that binds to TLR5. Additionally, it is unknown whether the TLR5 is upregulated by LPS. Further research into the upregulation of TLR5 is warranted. These results provide insight into immune responses and associated pathways contributing to the immune system in the liver during columnaris infection and induced response to LPS in largemouth bass.