The incidence of hepatocellular carcinoma (HCC) is rapidly increasing due to the prevalence of obesity and non-alcoholic fatty liver disease, but the molecular triggers that initiate disease development are not fully understood. We demonstrate that mice with targeted loss-of-function point mutations within the AMP-activated protein kinase (AMPK) phosphorylation sites on acetyl-CoA carboxylase 1 (ACC1 Ser79Ala) and ACC2 (ACC2 Ser212Ala) have increased liver de novo lipogenesis (DNL) and liver lesions. The same mutation in ACC1 also increases DNL and proliferation in human liver cancer cells. Consistent with these findings, a novel, liver-specific ACC inhibitor (ND-654) that mimics the effects of ACC phosphorylation inhibits hepatic DNL and the development of HCC, improving survival of tumor-bearing rats when used alone and in combination with the multi-kinase inhibitor sorafenib. These studies highlight the importance of DNL and dysregulation of AMPK-mediated ACC phosphorylation in accelerating HCC and the potential of ACC inhibitors for treatment. Display omitted •Fructose and ACC-activating mutations increase hepatic carcinogenesis•A liver-specific ACC inhibitor, ND-654, suppresses DNL and HCC proliferation•ND-654 reduces neutrophil recruitment and improves HCC survival•Humans with fatal HCC subtypes have increases in ACC expression Effective therapies are needed for treating hepatocellular carcinoma (HCC). Lally et al. report that fructose consumption and the genetic activation of acetyl-CoA carboxylase (ACC) increase hepatic de novo lipogenesis (DNL) and liver carcinogenesis. The liver-specific ACC inhibitor ND-654 is found to suppress hepatic DNL, inflammation, and HCC development.