AbstractBackgroundHepatocellular carcinoma (HCC) is a highly heterogeneous cancer with major challenges in both prevention and therapy. Metformin, adenosine monophosphate‐activated protein kinase (AMPK) activator, has been suggested to reduce the incidence of HCC when used for patients with diabetes in preclinical and clinical studies. However, the possible effects of metformin and their mechanisms of action in non‐diabetic HCC have not been adequately investigated.MethodsFah^−/− mice were used to construct a liver‐injury‐induced non‐diabetic HCC model for exploring hepatocarcinogenesis and therapeutic potential of metformin. Changes in relevant tumour and biochemical indicators were measured. Bulk and single‐cell RNA‐sequencing analyses were performed to validate the crucial role of proinflammatory/pro‐tumour CD8⁺ T cells. In vitro and in vivo experiments were performed to confirm Cyp26a1‐related antitumour mechanisms of metformin.ResultsRNA‐sequencing analysis showed that chronic liver injury led to significant changes in AMPK‐, glucose‐ and retinol metabolism‐related pathways in Fah^−/− mice. Metformin prevented the formation of non‐diabetic HCC in Fah^−/− mice with chronic liver injury. Cyp26a1 ddexpression in hepatocytes was significantly suppressed after metformin treatment. Moreover, downregulation of Cyp26a1 occurred in conjunction with increased levels of all‐trans‐retinoic acid (atRA), which is involved in the activation of metformin‐suppressed hepatocarcinogenesis in Fah−/− mice. In contrast, both CD8⁺ T‐cell infiltration and proinflammatory/pro‐tumour cytokines in the liver were significantly upregulated in Fah^−/− mice during chronic liver injury, which was notably reversed by either metformin or atRA treatment. Regarding mechanisms, metformin regulated the decrease in Cyp26a1 enzyme expression and increased atRA expression via the AMPK/STAT3/Gadd45β/JNK/c‐Jun pathway.ConclusionsMetformin inhibits non‐diabetic HCC by upregulating atRA levels and downregulating CD8⁺ T cells. This is the first reporting that the traditional drug metformin regulates the metabolite atRA via the Cyp26a1‐involved pathway. The present study provides a potential application of metformin and atRA in non‐diabetic HCC.