Liver fibrosis is a major consequence of chronic liver disease, where excess extracellular matrix is deposited, due caused by the activation of hepatic stellate cells (HSCs). The suppression of collagen production in HSCs is therefore regarded as a therapeutic target of liver fibrosis. The present study investigated effects of harmine, which is a β-carboline alkaloid and known as an inhibitor of dual-specificity tyrosine-regulated kinases (DYRKs), on the production of collagen in HSCs. LX-2 cells, a human HSC cell line, were treated with harmine (0–10 μM) for 48 h in the presence or absence of TGF-β1 (5 ng/ml). The expression of collagen type I α1 (COL1A1) and DYRK isoforms was investigated by Western blotting, quantitative RT-PCR, or immunofluorescence. The influence of knockdown of each DYRK isoform on the COL1A1 expression was further investigated. The expression of COL1A1 was markedly increased by treating with TGF-β1 for 48 h in LX-2 cells. Harmine (10 μM) significantly inhibited the increased expression of COL1A1. LX-2 cells expressed mRNAs of DYRK1A, DYRK1B, DYRK2, and DYRK4, although the expression of DYRK4 was much lower than the others. Knockdown of DYRK1B, but not DYRK1A or DYRK2, with siRNA significantly suppressed TGF-β1-induced increase in COL1A1 expression. These results suggest that harmine suppresses COL1A1 expression via inhibiting DYRK1B in HSCs and therefore might be effective for the treatment of liver fibrosis. •Harmine suppressed the production of collagen induced by TGF-β1 in LX-2 cells.•LX-2 cells expressed mRNAs of DYRK1A, DYRK1B, and DYRK2.•DYRK1B knockdown suppressed the production of collagen 1A1 induced by TGF-β1 in LX-2 cells.