Aims/hypothesis Despite the current pandemic of metabolic diseases, our understanding of the diverse nature of the development of metabolic alterations in people who eat a high-fat diet (HFD) is still poor. We recently demonstrated a cardio-metabolic adaptation in mice fed a HFD, which was characterised by a specific gut and periodontal microbiota profile. Since the severity of hepatic disease is characterised by specific microRNA (miRNA) signatures and gut microbiota is a key driver of both hepatic disease and miRNA expression, we analysed the expression of three hepatic miRNA and studied their correlation with hepatic triacylglycerol content and gut microbiota.Methods A cohort of C57BL/6 4-week-old wild-type (WT) male mice (n=62) was fed a HFD for three months to provide a model of metabolic adaptation. 8-week-old C57BL/6 mice, either WT or of different genotypes and with different or no gut microbiota (axenic, ob/ob, Nod1, Cd14 knockout [Cd14KO] and Nod2) were fed a normal chow. Then, glycaemic index, body weight, blood glucose levels and hepatic triacylglycerols were measured. Gut (caecum) microbiota taxa were analysed by pyrosequencing. To analyse hepatic miRNA expression, real-time PCR was performed on total extracted miRNA samples. Data were analysed using two-way ANOVA followed by the Dunnett’s post hoc test, or by the unpaired Student’s t test. A cluster analysis and multivariate analyses were also performed.Results Our results demonstrated that the expression of miR-181a, miR-666 and miR-21 in primary murine hepatocytes is controlled by lipopolysaccharide in a dose-dependent manner. Of the gut microbiota, Firmicutes were positively correlated and Proteobacteria and Bacteroides acidifaciens were negatively correlated with liver triacylglycerol levels. Furthermore, the relative abundance of Firmicutes was negatively correlated with hepatic expression of miR-666 and miR-21. In contrast, the relative abundance of B. acidifaciens was positively correlated with miR-21.Conclusions/interpretation We propose the involvement of hepatic miRNA, liver triacylglycerols and gut microbiota as a new triad that underlie the molecular mechanisms by which gut microbiota govern hepatic pathophysiology during metabolic adaptation to a HFD.