To investigate the effects of varying NAT1 activity in different cell lines, mitochondrial oxidative phosphorylation, aerobic glycolysis and mitochondrial fuel usage was quantified in a panel of human cell lines. As NAT1 activity increased, mitochondrial reserve respiratory capacity increased while aerobic glycolysis decreased. In addition, phosphorylation of PDH-E1α in these cells limited their ability to use glucose as a primary fuel source. Those cells with high NAT1 activity exhibited a quiescent metabolic phenotype and proliferated more slowly. This might explain, in part, why some cancer patients with low NAT1 expression in their tumour tissue show poorer survival outcomes compared to those with high NAT1 expression. The current study demonstrated that NAT1 enzymatic activity is important for metabolism in cancer cell lines and increasing NAT1 activity may better equip cells to survive under stressed conditions by increasing reserve respiratory capacity.