<p>As a cytoskeleton component and one of the most abundant and conserved proteins in eukaryotic cells, actin involves functions including growth, differentiation, and mechanical stability. However, the role of actin cytoskeleton in mitochondrial energy homeostasis and cell signalling status in the brain and gut axis is uncertain in fish. We, thus, examined the in vitro effect of cytochalasin D (Cyt D), an inhibitor of actin polymerisation/depolymerisation, to explore the role of actin disruption in regulating mitochondrial energy metabolism and cell signal status of the brain/gut axis of air-breathing fish (<i>Anabas testudineus</i> Bloch). Increasing doses of Cyt D (10⁻⁹&#xa0;M, 10⁻⁸&#xa0;M, 10⁻⁷&#xa0;M) were incubated with brain/gut segments for 15&#xa0;min. The activity pattern of mitochondrial energetic markers such as cytochrome C oxidase (COX), succinate dehydrogenase (SDH) and cytosolic marker lactate dehydrogenase (LDH), and the status of ROS and NO were quantified in the brain segments such as prosencephalon (PC), mesencephalon (MC) and mesencephalon (MeC) and the gut segments like anterior (AI), mid (MI) and posterior intestines (PI). Graded doses of Cyt D boosted the COX activity in all segments except PI, which showed a fall. After Cyt D exposure, the SDH and LDH activities increased in the MC, MeC and AI but decreased in the PC, MI and PI segments. Cyt D exposure produced a rise in ROS in MeC, MI and AI but decreased in PC, MC and PI segments. Cyt D lowered the NO content in all brain and gut segments except PI, where it dropped. The data indicate an inverse relationship between COX and NO in the brain/gut axis. Similarly, upon actin disruption, a direct synergistic interaction occurs between NO and ROS in the brain segments, but an inverse antagonistic interaction operates between them in gut segments. Overall, the data provide evidence for a critical differential role for actin in regulating mitochondrial energetics, ROS, and NO status across the brain/gut axis.</p>