Liver fibrosis is a significant consequence of chronic liver diseases, where excess deposition of extracellular matrix is caused by the activation of hepatic stellate cells (HSCs). The suppression of HSC activation is therefore regarded as a therapeutic target of liver fibrosis. The present study investigated the involvement of protein arginine methyltransferase 5 (PRMT5), which mediates genome organization and cell cycle regulation, in HSC activation. LX-2 cells, a human HSC cell line, were treated with TGF- β1 for 48 h in the presence of PRMT5 inhibitors (EPZ015666 and JNJ64619178). The expression of α-smooth muscle actin (α-SMA) and type I collagen α1 (COL1A1), activated HSC markers, were markedly increased by the TGF-β1 treatment. PRMT5 inhibitors suppressed the increased expression of α-SMA and COL1A1 in a concentration-dependent manner. Knockdown of PRMT5 also suppressed the TGF-β1-induced COL1A1 expression in LX-2 cells. RNA-sequencing analysis showed that GO terms related to ECM production and SMAD signaling were enriched with RNA of LX-2 cells treated with the PRMT5 inhibitor JNJ64619178. These results suggest that PRMT5 promotes HSC activation, possibly depending on the SMAD signaling pathway, and therefore might be a target for the prevention and treatment of liver fibrosis.