Amauroderma rugosum (Blume & T. Nees) Torrend (Ganodermataceae) is an edible mushroom with a wide range of medicinal values. Our previous publication demonstrated the therapeutic effects of the water extract of A. rugosum (WEA) against gastric ulcers. However, the protective effects of the ethanol extract of A. rugosum (EEA) on gastric mucosa and its major active constituents have not yet been elucidated.This study aims to evaluate the gastroprotective effects and underlying mechanisms of EEA and its fat-soluble constituent, ergosterol, in acute gastric ulcers.SD rats were pre-treated with EEA (50, 100, and 200 mg/kg) or ergosterol (5, 10, and 20 mg/kg), and acute gastric ulcer models were constructed using ethanol, gastric mucus secretion inhibitor (indomethacin) or pyloric-ligation. The gastric ulcer area, histological structure alterations (H&E staining), and mucus secretion (AB-PAS staining) were recorded. Additionally, Q-PCR, western blotting, immunohistochemistry, ELISA, molecular docking, molecular dynamics simulations, MM-GBSA analysis, and surface plasmon resonance assay (SPR) were used to investigate the underlying mechanisms of the gastroprotective effect.Compared with WEA, which primarily exerts its anti-ulcer effects by inhibiting inflammation, EEA containing fat-soluble molecules showed more potent gastroprotective effect through the promotion of gastric mucus secretion, as the anti-ulcer activity was partly blocked by indomethacin. Meanwhile, EEA exhibited anti-inflammatory effects by suppressing the production of IL-6, IL-1β, TNF-α, and NO, thereby inhibiting the MAPK pathway. Significantly, ergosterol (20 mg/kg), the bioactive water-insoluble compound in EEA, exhibited a gastroprotective effect comparable to that of lansoprazole (30 mg/kg). The promotion of gastric mucus secretion contributed to the effects of ergosterol, as indomethacin can completely block it. The upregulations of COX1-PGE2 and C-fos, an activator protein 1 (AP-1) transcription factor, were observed after the ergosterol treatment. Ergosterol acted as an LXRβ agonist via van der Waals binding and stabilizing the LXRβ protein without compromising its flexibility, thereby inducing the upregulation of AP-1 and COX-1.EEA and its primary bioactive compound, ergosterol, exert anti-ulcer effects by promoting gastric mucus secretion through the LXRβ/C-fos/COX-1/PGE2 pathway.Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.