NAD+ is a ubiquitous metabolite that plays a critical role in the regulation of many metabolic pathways. Recent work has shown that boosting NAD+ biosynthesis can protect against the development of hepatocellular carcinoma. Here we have explored whether organelle specific changes in NAD+ are important for this effect. We have examined transgenic animals overexpressing either NMNAT1 or NMNAT3 to increase NAD+ biosynthesis in the nuclear and mitochondrial compartments respectively. The transgenic mice and WT littermates were injected with the liver carcinogen diethylnitrosamine (DEN) at 2 weeks of age and subsequently placed on a high-fat diet to accelerate tumourigenesis. Overexpression of NMNAT1 significantly reduced the multiplicity of liver tumours, with an average of 5 tumours per mouse, compared to 13 in WT. In contrast, NMNAT3 overexpression did not influence tumour multiplicity. However when tumours were established the transgenic models showed no protection with regards to tumour burden. The mice with higher nuclear NAD+ biosynthesis displayed a 50% reduction in serum levels of the liver damage marker ALT compared to WT littermates. We postulated that increased nuclear NAD+ levels may be important in protecting against tumour initiation events and to test this further we treated 12-16 week old transgenic and WT littermates with an acute dose of DEN. NMNAT1 transgenic mice had a 3-fold lower ALT response compared WT and NMNAT3 transgenic mice, indicating that nuclear NAD+ is protective against genotoxic liver damage. Our study shows that the subcellular changes in NAD+ are important for protection against liver tumour formation.