The steroid hormone cortisol is released as the final product of the stress response in teleost fish and is essential in aiding survival. When an animal perceives a stressor, substantial amounts of cortisol are produced and released into the blood, reallocating resources to various tissues and improving the response to the stressor. While high plasma cortisol levels can be beneficial for short durations, prolonged elevated levels of cortisol, produced in response to a chronic stressor such as captivity, can negatively impact various functions including growth and reproduction. The effect of long-term captivity on interrenal physiology was investigated in pubertal New Zealand shortfin eels (Anguilla australis). Wild-caught fish were held in tanks for up to eight months, with periodic terminal sampling of individuals at different timepoints. Plasma cortisol levels were compared between groups and the interrenal physiology of eels was analysed by measuring the nuclear diameters of the interrenal cells. Immunoreactivity of the interrenal cells to key enzymes involved in cortisol biosynthesis, cytochrome P450 side-chain cleavage (Cyp11a1) and 11β-hydroxylase (Cyp11b), was also analysed. As captivity length progressed, a significant reduction in immunoreactivity for interrenal tissues stained for either Cyp11a1 or Cyp11b was observed, alongside a concurrent decrease in interrenal nuclear diameters. Unusually, plasma cortisol levels did not differ significantly between the groups, suggesting a potential reduction in cortisol metabolism or increase of cortisol-binding globulin in the plasma to compensate for the apparent reduction in interrenal activity. This study provides valuable preliminary information on the impacts of chronic stress on interrenal physiology and localised cortisol production in A. australis. As chronic stress is known to negatively impact various tissues and functions, including reproduction, understanding the impacts of long-term captivity as a chronic stressor is vital to further aquacultural development of fish such as the shortfin eel.