DHCR24 (24-dehydrocholesterol reductase), or Seladin-1, is one of the most expressed genes in the adrenal gland. Since the rat and human adult adrenal cortex differ in their respective functional properties, the aim of the present study was to verify whether Seladin-1 may be differentially involved in basal and ACTH-stimulated steroidogenesis and oxidative stress management. Seladin-1 expression was predominantly observed in both human and rat zona fasciculata, with a predominant cytoplasmic localization in human cells and a nucleo-cytoplasmic distribution in rat cells. In human fasciculata cells, localization of the protein was primarily associated with the endoplasmic reticulum (ER). While its expression was increased by ACTH, its intracellular localization was not altered by ACTH treatment (10 nM) or by the Seladin-1 inhibitor U18666A (75 nM). Pre-incubation with U18666A did not modify the ACTH-induced increase in cortisol secretion, but abolished the ACTH-induced increase in DHEA secretion. In rat fasciculata cells, ACTH induced a massive redistribution of Seladin-1 from the cytoplasm (cis-Golgi apparatus) to the nucleus, which was inhibited by pre-incubation with U18666A. Pre-incubation with U18666A also decreased ACTH-induced Seladin-1 and 11-hydroxylase protein expression as well as corticosterone production, increased ACTH-induced ROS production but decreased ACTH-induced expression of the detoxifying protein Akr1b7. Thus, protection against acutely elevated ACTH-induced oxidative stress in rat fasciculata cells is correlated with nuclear relocalization of Seladin-1 and its effects on cellular detoxifying machinery. Altogether, these results indicate that Seladin-1 expression and intracellular localization are correlated with both the intensity and nature of ACTH-induced steroidogenesis and resultant oxidative stress.