Nicotinamide nucleotide transhydrogenase (NNT) is a highly conserved inner mitochondrial membrane protein, which supplies high concentrations of NADPH for detoxification of reactive oxygen species (ROS) by glutathione and thioredoxin pathways. In humans, loss-of-function mutations in NNT cause familial glucocorticoid deficiency, a potentially fatal, adrenal specific disorder characterized by increased levels of ACTH and low levels of cortisol. Nnt−/− mice (MUT) have a 50% reduction in both basal and ACTH-stimulated corticosterone output when compared to their WT counterparts (WT). The reintroduction of Nnt into this mouse strain by a BAC transgene (BAC) results in 1.9-fold overexpression of the protein and partially rescues the phenotype. RNA-seq on adrenals from WT, MUT and BAC mice revealed differential expression (fold change ≧1.5; P value <0.001) of 91 genes between WT and MUT mice that was reversed in the BAC suggesting these are directly affected by Nnt loss. The 91 genes fell into 12 biological processes by gene ontology analysis with significant enrichment (4.13-fold; P<0.05) of genes involved in stress response including the heat shock proteins Dnajb1, Hsph1, Hspa1a and Hspa1b. Interestingly alpha- and beta-haemoglobins (Hba-a1, Hba-a2, Hbb-b1 and Hbb-b2) were highly upregulated in the knockout mouse and their levels returned to normal in the BAC rescue. This data suggest that ROS damage to proteins is activating mito- and cytoprotective proteins (haemoglobins and heat shock proteins respectively) that help maintain cell viability. Surprisingly no alterations in antioxidant genes of the glutathione and thioredoxin pathways (Prdx3, Gpx1, Sod2, Txnrd2, Gr) or in components of the ACTH signalling pathway (Mc2r, Mrap) were noted, whereas a 25% down-regulation, at RNA level, in mitochondrial steroidogenic enzymes (Cyp11a1, Cyp11b1) resulted in a 60% reduction at protein level, perhaps providing a mechanism to explain the reduction in corticosterone seen in the mice.