Abstract Adrenocortical carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. In this study, we evaluated a potential new treatment target for ACC, focusing on the mitochondrial reduced form of NAD phosphate (NADPH) generator nicotinamide nucleotide transhydrogenase (NNT). NNT has a central role within mitochondrial antioxidant pathways, protecting cells from oxidative stress. Inactivating human NNT mutations result in congenital adrenal insufficiency. We hypothesized that 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. As predicted, this manipulation increased intracellular levels of oxidative stress; this resulted in a pronounced suppression of cell proliferation and higher apoptotic rates, as well as sensitization of cells to chemically induced oxidative stress. Steroidogenesis was paradoxically stimulated by NNT loss, as demonstrated by mass spectrometry–based steroid profiling. Next, we generated a stable NNT knockdown model in the same cell line to investigate the longer lasting effects of NNT silencing. After long-term culture, cells adapted metabolically to chronic 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 these responses were explored in detail by RNA sequencing and nontargeted metabolome analysis, revealing major alterations in nucleotide synthesis, protein folding, and polyamine metabolism. This study provides preclinical evidence of the therapeutic merit of antioxidant targeting in ACC as well as illuminating the long-term adaptive response of cells to oxidative stress.