Increased sugar consumption promotes excess fat synthesis in the liver through de novo lipogenesis, a hallmark of metabolic dysfunction-associated fatty liver disease (MAFLD)1. MAFLD is a rising cause of liver cancer. Acetyl-CoA carboxylase 1 (ACC1) plays a major role in fat synthesis, while acetyl-CoA carboxylase 2 (ACC2) regulates fat breakdown. Both are potential targets for liver cancer therapy2. In our previous study, transgenic mice with liver-specific knockout of ACC1 and ACC2 genes, administered with diethylnitrosamine (DEN) and fed a high-fat diet, showed an unexpected increase in liver tumours compared to the wild-type (WT)3. In this study, we investigated how selective inhibition of ACC isotypes in the liver, the timing of ACC inhibition, and a standard chow diet impact liver tumorigenesis. We used six genotypes of female C57BL/6 mice with floxed ACC1 and/or ACC2 alleles. The mice were injected with DEN at 2 weeks of age, and liver-specific deletion of ACC genes was achieved with AAV8-TBG-Cre virus injection at 9 weeks of age. The mice were fed a standard chow diet and euthanised at 52 weeks of age and livers were examined for tumours. We found that mice completely lacking both ACC1 and ACC2 in the liver had a greater than 5-fold increase in tumour phenotypes compared to the wild-type. In contrast, deletion of ACC1 or ACC2 alone had no significant impact on tumour phenotypes. Our study confirms that increased tumour phenotypes are observed in a DEN liver cancer mouse model with complete inhibition of both ACC isotypes in the liver, regardless of diet, timing, and gene deletion method