MicroRNA-21 (miR-21) has emerged as a critical regulatory molecule and an important serum marker in hepatic fibrogenesis. The aim of the present study was to investigate the role of inhibiting miR-21 on hepatic fibrosis treatment. Serum miR-21 levels in 60 healthy individuals and 180 patients with different stages of liver cirrhosis were examined, miR-21 levels in normal or cirrhotic human liver tissues (n = 10 each) were also detected. An adenoviral vector (Ad-TuD-21) carrying the sponging ToughDecoy (TuD)-RNA sequence against miR-21 was constructed to reduce miR-21 expression efficiently in vitro and in vivo. Histological and immunohistological examinations were performed to evaluate the inhibitory effects and mechanism of Ad-TuD-21 delivery into carbon tetrachloride (CCl4) induced hepatic fibrosis rats by targeting extracellular signal-regulated kinase 1 (ERK1) signalling in hepatic stellate cells (HSC) and hepatocyte epithelial-mesenchymal transition (EMT). Our results revealed that enhanced miR-21 levels in cirrhotic patients were related to the severity and activity of liver cirrhosis. Ad-TuD-21 administered to liver fibrosis rats could remarkably suppress profibrotic gene expression, cause histological improvements in liver and attenuate hepatic fibrosis significantly. More importantly, after Ad-TuD-21 treatment, inhibition of both the ERK1 signalling pathway in HSC and hepatocyte EMT was confirmed, which paralleled the enhancement of miR-21 target genes - sprouty2 (SPRY2) and hepatocyte nuclear factor 4 alpha (HNF4 alpha) - expression in vivo. These data demonstrated that miR-21 is a key regulator to promote hepatic fibrogenesis, and sponging miR-21 expression may present a novel potentially therapeutic option for hepatic fibrosis.