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Aims: Biliary diseases represent around 10% of all chronic liver diseases and affect both adults and children. Currently available biochemical tests detect cholestasis but not early liver fibrosis. Circulating extracellular vesicles (EVs) provide a noninvasive, real-time molecular snapshot of the injured organ. We thus aimed at searching for a panel of EV-based biomarkers for cholestasis-induced early liver fibrosis using mouse models. Results: Progressive and detectable histological evidence of collagen deposition and liver fibrosis was observed from day 8 after bile duct ligation (BDL) in mice. Whole transcriptome and small RNA sequencing analyses of circulating EVs revealed differentially enriched RNA species after BDL versus sham controls. Unsupervised hierarchical clustering identified a signature that allowed for discrimination between BDL and controls. In particular, 151 microRNAs (miRNAs) enriched in BDL-derived EVs were identified, of which 66 were conserved in humans. The liver was an important source of circulating EVs in BDL animals as evidenced by the enrichment of several hepatic mRNAs, such as Albumin and Haptoglobin . Interestingly, among experimentally validated miRNAs, miR192-5p, miR194-5p, miR22-3p, and miR29a-3p showed similar enrichment patterns also in EVs derived from 3,5-diethoxycarboncyl-1,4-dihydrocollidine-treated (drug-induced severe cholestasis) but not in mice with mild phenotype or non-cholestatic liver fibrosis. Innovation: A panel of mRNAs and miRNAs contained in circulating EVs, when combined, indicates hepatic damage and fibrosis in mice and represents promising biomarkers for human severe cholestasis-induced liver fibrosis. Conclusion: Analysis of EV-based miRNAs, in combination with hepatic injury RNA markers, can detect early cholestatic liver injury and fibrosis in mice. Antioxid. Redox Signal. 36, 480-504.