Nicotinamide nucleotide transhydrogenase (NNT) is a NADPH-generating mitochondrial proton pump with a central role in mitochondrial antioxidant pathways. Recent studies revealed inactivating NNT mutations in patients with familial glucocorticoid deficiency, indicating a selective susceptibility of the adrenal cortex to NNT deficiency and oxidative stress. Here we explored the potential value of NNT as a therapeutic target in adrenocortical cancer. We delineated the distinct effects of NNT loss on cellular proliferation and steroidogenesis, employing two in vitro knockdown models, including transient siRNA-mediated knockdown and shRNA-mediated stable loss of NNT in the adrenocortical cell line NCI-H295R. Transient NNT knockdown impaired cellular redox balance, resulting in a lower ratio of reduced to oxidised glutathione on luminescence-based quantification. Assessment of proliferation using a fluorescent DNA dye revealed a loss in cellular viability and decrease in proliferation with NNT knockdown (64±8% decrease compared to scrambled siRNA-transfected cells, p<0.01). NNT-deficient cells also became exceedingly sensitive to mild chemically induced oxidative stress. After long-term culture, H295R cells with stable NNT knockdown appeared to develop compensatory mechanisms, improving their redox balance and proliferative potential. This adaptation was associated with alterations in glycolytic and oxygen consumption rates, as demonstrated by extracellular flux analysis using Seahorse XF technology. Steroid profiling by liquid chromatography-tandem mass spectrometry revealed a distinct profile induced by transient NNT knockdown, comprising lower 17OH-progesterone but, surprisingly, higher androstenedione and cortisol synthesis, with a pronounced increase in 11β-hydroxylase activity (ratio of 11-deoxycortisol/cortisol 93±16 in cells transfected with scrambled siRNA vs. 58±8 with NNT knockdown, p<0.01). A similar steroidogenic pattern was observed with stable knockdown. Our study suggests a potential role of NNT inhibition as a novel therapeutic approach in advanced adrenocortical carcinoma. Steroid profiling reveals a surprising increase in glucocorticoid and androgen synthesis with NNT loss, challenging the previous association of impaired redox balance and adrenal insufficiency.