Regulatory volume decrease (RVD) constitutes a fundamental process that turbot (Scophthalmus maximus) hepatocytes are able to perform when exposed to hypo-osmotic stress. RVD is an integrative mechanism that involves various elements of the cellular machinery. Among others, ATP is an essential protagonist: released following hypo-osmotic shock, it acts as an auto/paracrine factor to trigger other signalling events. The origin of this ATP remains unclear and, to the best of our knowledge, no information exists about the role of mitochondrial respiration in RVD. Therefore, we propose to analyse the potential link between RVD and the respiratory chain, with a focus on ATP release and exocytosis. Using inhibitors of mitochondrial respiration, RVD was shown to be dependent on respiratory chain activity. However, we demonstrated an indirect role of mitochondrial respiration: ATP could be synthesized and then stored in intracellular vesicles until the moment cells release it to face hypo-osmotic swelling. However, the involvement of exocytosis in this process needs to be further investigated.