Recent studies suggest that the important xenobiotic metabolizing enzyme, human arylamine N-acetyltransferase 1 (NAT1), may be important in cancer cell invasion and metastasis (Tiang et al., 2015). Matrix metalloproteinases (MMPs) are an important family of zinc-dependent endo-proteinases that degrade the extracellular matrix and promote cell invasion. To investigate its role in cancer cell invasion, NAT1 was knocked out in the highly invasive triple negative breast cancer cell line MDA-MB-231 using CRISPR technology. The aim of this study was to determine the effect of NAT1 knockout on the ability of MDA-MB-231 cells to invade in vitro and to determine the underlying mechanism for altered MMP gene expression. Invasion and migration assays were performed using the ACEA xCELLigence system and Matrigel-coated membranes. Invadopodia degradation assays were performed on fluorescein-conjugated gelatin-coated coverslips and visualised by confocal microscopy. MMP gene expression was quantified by real-time RT-PCR and protein expression in cells and growth medium was determined by Western blot. Although cell migration was not different, NAT1 KO cells had a reduced ability to invade compared to parent cells. The gene expression of some MMPs changed significantly upon NAT1 knockout, with MMPs 2, 7, 9 and 14 all increasing, while MMP1 decreased. Western blot of culture medium from NAT1 KO cells showed that MMP9 was significantly increased compared to parent cells, while MMP1 was significantly decreased compared to parent cells. In addition, treatment of parent cells with the histone deacetylase inhibitor trichostatin A indicated that these changes may be associated with altered histone acetylation. Since NAT1 is unable to acetylate proteins itself, further studies are required to determine the mechanism by which loss of NAT1 leads to changes in MMP gene expresson.