NADPH is an important cofactor in many biosynthesis pathways that control fundamental cellular processes. We recently determined the crystal structure of HSCARG, with functions previously unknown, and demonstrated it is an NADPH sensor, which undergoes restructuring and redistribution in response to changes of intracellular NADPH/NADP levels. In this study, we identified argininosuccinate synthetase (AS), a rate-limiting enzyme in nitric oxide synthesis, as capable of associating with HSCARG and demonstrated further that HSCARG decreased nitric oxide synthesis by down-regulating AS activity, whereas AS overexpression up-regulated hscarg mRNA transcription, suggesting a negative feedback mechanism. A decrease in the NADPH/NADP(+) ratio, induced by dehydroepiandrosterone treatment, enhanced the interaction between HSCARG and AS, which resulted in stronger inhibition of AS activity and nitric oxide production. The dimerization region of HSCARG, amino acids 153-189, was identified to undergo critical interactions with AS. Furthermore, the viability of HSCARG RNA interference-treated epithelial cells decreased significantly, accompanied by an increase of the activity of caspase-3, which suggested that the loss of viability was because of apoptosis. These results indicate that HSCARG regulation of AS activity is crucial for maintaining the intracellular balance between redox state and nitric oxide levels.