The human arylamine N-acetyltransferases are a family of two (NAT1 & 2) Phase II xenobiotic-metabolising enzymes that acetylate heterocyclic amine, hydrazine and arylamine substrates. Recently, NAT1 expression has been linked to breast cancer survival, with a positive correlation found between tumour NAT1 protein expression and five-year survival rate in patients [1]. This result correlates with a number of microarray studies that found decreased NAT1 mRNA expression in chemo-resistant cell lines [2, 3]. In this study, we show that the CRISPR-mediated knockout of NAT1 in breast cancer cells (MDA-MB-231 & T-47D) resulted in significant resistance to drug-induced cell death (cisplatin, epirubicin, daunorubicin, paclitaxel, vincristine and 5-fluoruracil) independent of their mechanism of action. Moreover, cells were treated with the oxidant tert-butyl hydrogen peroxide demonstrated similar resistance. Drug sensitivity in the NAT1-deleted cells was rescued with the stable re-insertion of NAT1 expressed using a pcDNA3 vector, indicating that the observed resistance was not due to off-target effects. The NAT1 KO cells showed significant reduction in release of mitochondrial cytochrome c in response to all tested drugs suggesting a dysfunctional intrinsic apoptotic pathway. Inhibition of autophagy sensitised the KO cells so their response to chemotherapy (cisplatin) was similar to parental cells. These results provide a mechanism that explains chemo-resistance in breast cancer patients with low tumour NAT1 expression.