Recent investigations have indicated that the arylamine N-acetyltransferase 1 enzyme (NAT1) plays an important role in cancer cell biology. Moreover, factors that regulate NAT1 activity can influence cell growth, survival, and metastatic potential. Preliminary data from our laboratory showed that NAT1 expression affects the acetylation of intracellular proteins including the sirtuins SIRT1 and SIRT2, which control mitochondrial biogenesis and stress resistance. We therefore hypothesised that NAT1 may be important in the regulation of cancer cell metabolism. In the present study, we knock out NAT1 using CRISPR technology and demonstrate that loss of NAT1 decreases mitochondrial respiration in human cancer cells via alteration in the pyruvate dehydrogenase complex (PDC). Inhibition of NAT1 leads to inactivation of PDC either via phosphorylation of the pyruvate dehydrogenase α subunit or reduction of its expression, in a cell-dependent manner. Loss of NAT1 decreases tumor cell proliferation and induces apoptotic cell death under glucose deprivation as a result of oxidative stress. Loss of NAT1 also decreases acetylation of p53 as well as AKT phosphorylation, which contributes to cell apoptosis when nutrients are deprived. Since nutrient deprivation occurs commonly in solid tumours as a result of poor blood supply, our findings indicate that NAT1 may be important in modulating cell survival under these conditions and could be a potential drug target for certain cancers.