Liver fibrosis (LF) is a pathological process of the liver that threatens human health. Currently, effective treatments are still lacking. Esculin, a prominent constituent found in the Fraxinus rhynchophylla. (bark), Aesculus hippocastanum. (bark), and Cichorium intybus. (herb), has been shown to possess significant anti-inflammatory, antioxidant, and antibacterial properties. However, to date, there have been no studies investigating its potential efficacy in the treatment of LF.The study aims to investigate the therapeutic effect of esculin on LF and elucidate its potential molecular mechanism.Carbon tetrachloride (CCl4) was injected intraperitoneally to induce LF in mice, and transforming growth factor β1 (TGF-β1) was injected to induce LX-2 cells to investigate the improvement effect of esculin on LF. Kit, histopathological staining, immunohistochemistry (IHC), immunofluorescence (IF), polymerase chain reaction (PCR), and western blot (WB) were used to detect the expression of fiber markers and nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling pathway in liver tissue and LX-2 cells. Finally, molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) were used to verify the targeting between Nrf2 and esculin.Esculin significantly inhibited CCl4-induced hepatic fibrosis and inflammation in mice. This was evidenced by the improvement of liver function indexes, fibrosis indicators, and histopathology. Additionally, esculin treatment prominently reduced the levels of pro-inflammatory factors, oxidative stress, and liver Fe2+ in CCl4-induced mice. In vitro studies also showed that esculin treatment significantly inhibited TGF-β1-induced LX-2 cell activation and decreased alpha-smooth muscle actin (α-SMA) and collagen I expression. Mechanism experiments proved that esculin can activate the Nrf2/GPX4 signaling pathway and inhibit liver ferroptosis. However, when LX-2 cells were treated with the Nrf2 inhibitor (ML385), the therapeutic effect of esculin significantly decreased.This study is the first to demonstrate that esculin is a potential natural active ingredient in the treatment of LF, which can inhibit the activation of hepatic stellate cells (HSC) and improve LF. Its therapeutic effect is related to the activation of the Nrf2/GPX4 signaling pathway.Copyright © 2024. Published by Elsevier GmbH.