Adrenocortical Carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. Here we evaluated a potential new treatment target for ACC, focusing on the mitochondrial NADPH generator Nicotinamide Nucleotide Transhydrogenase (NNT). NNT has a central role within the mitochondrial antioxidant pathways, protecting cells from oxidative stress. Inactivating NNT mutations lead to isolated primary adrenal insufficiency, suggesting a selective vulnerability of adrenocortical cells to NNT loss. A TCGA database search confirmed increased NNT expression in ACC. Therefore, we hypothesised NNT silencing in ACC cells will induce toxic levels of oxidative stress. To explore this, we transiently knocked down NNT in NCI-H295R ACC cells by siRNA transfection. NNT silencing increased intracellular levels of oxidative stress; this resulted in a dramatic suppression of cell proliferation and higher apoptotic rates, as well as sensitising cells to chemically-induced oxidative stress. Steroidogenesis was paradoxically stimulated by NNT loss, as demonstrated by comprehensive steroid profiling. Next, we generated a stable NNT knockdown model in the same cell line (lentiviral shRNA transfection), to understand the chronic effects of NNT silencing. After culture for 13 months, cells adapted metabolically to stable NNT knockdown, restoring their redox balance and resilience to oxidative stress, although their proliferation remained suppressed. This was associated with higher rates of oxygen consumption. The molecular pathways underpinning the cellular response to transient and chronic NNT loss were explored in detail by RNA sequencing and whole-metabolome analysis. Transient NNT knockdown led to changes in core pathways controlling cellular proliferation and viability. Stable (chronic) knockdown was characterised by changes consistent with accelerated protein turnover and up-regulation of antioxidant polyamines, which can facilitate partial adaptation to oxidative stress. Our study provides the first pre-clinical evidence of the therapeutic merit of antioxidant targeting in ACC, as well as delineation of the long-term adaptive response of cells to oxidative stress.