While tissue-specific immune responses are well-understood in mammals, such studies are lacking for marine bivalves. In this study, we investigated immune responses in gill, hepatopancreas and haemolymph of mussels (Perna canaliculus Gmelin, 1791) following experimental infection with Vibrio sp. DO1 (V. coralliilyticus/neptunius-like isolate). Significant differences in metabolite profiles and metabolic responses between tissues were observed. Overall, haemolymph and gills shared common metabolic responses characterized by increases in itaconic acid and decreases in other amino acids (e.g., BCAAs, lysine, tryptophan) and fatty acids (e.g., DHA, EPA, palmitoleic acid). Increases in itaconic acid, decreases in fatty acids, and increases in amino acids were found in hepatopancreas tissues. The alterations of these metabolites suggest osmotic stress, oxidative stress, changes in amino acid metabolism and protein synthesis in the immune system of P. canaliculus caused by Vibrio sp. infection. Interestingly, the accumulation of itaconic acid in all three tissues of infected mussels suggests that this metabolite has an important role in the mediation of bivalve antimicrobial activities and immune responses. These results indicate that careful consideration should be given to tissue sampling choices for immunological and metabolomics studies. In addition, further investigations are needed to elucidate mechanistic responses across different tissues associated with pathophysiological processes in bivalves.