MicroRNAs have emerged as essential regulators in the biological process of liver regeneration by modulating the post-transcriptional expression of the target genes. In the present study, we found miR-20a expression is decreased remarkably in three rodent liver regeneration models using miRNA PCR array and Venn diagram analysis. Inhibition of miR-20a expression enhanced hepatocytes proliferation in vivo and in vitro. In contrast, overexpression of miR-20a reduces hepatocytes proliferation and subsequently impaired liver regeneration in the mouse PHx model. Moreover, we have identified TCF4 as a target gene of miR-20a using the PCR Array and luciferase assay. Next, mice with TCF4 deficiency were used to establish the PHx model and subjected to the examination of liver regeneration capacity. We found TCF4-deficient mice exhibited impaired liver regeneration compared with control. Given that TCF4 acts as a transcription factor, we sort to elucidate the downstream genes involved in liver regeneration. Promoter analysis and Chip assay confirmed that TCF4 enhances CDC2 and CDC6 expression through binding to the promoter region and leads to the proliferation and cell cycle progression in hepatocytes. In conclusion, this study provides evidence that the miR20a-TCF4-CDC2/6 axis plays an essential role during liver regeneration.