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Background Liver fibrosis, a key transition from chronic liver disease to cirrhosis, is driven by hepatic stellate cell (HSC) activation and excessive extracellular matrix deposition. Enhanced aerobic glycolysis in HSCs supports their activation and fibrogenesis. The TGF-β/SMAD3 pathway is a central pro-fibrotic signal and also regulates glycolytic reprogramming in HSCs. Gastrodin, a natural compound, shows anti-fibrotic potential. This study explored its effects and whether it acts through dual regulation of TGF-β/SMAD3 signaling and glycolysis.Methods Activated human HSCs (LX2) were treated with gastrodin in vitro. Fibrosis markers, TGF-β/SMAD3 components, and glycolytic enzymes were assessed via qPCR/Western blot. Glycolytic flux was measured. In vivo, gastrodin was given to CCl 4-induced fibrotic mice. Liver pathology, fibrosis markers, pathway activity, and glycolysis were analyzed using histology, molecular assays, RNA-seq, and protein interaction studies.Results In vitro, gastrodin reduced HSC activation, downregulated fibrosis markers, and inhibited both TGF-β/SMAD3 signaling and glycolysis. In vivo, it attenuated liver fibrosis, suppressed the TGF-β/SMAD pathway, and decreased glycolytic activity. Multi-omics analyses confirmed that gastrodin targets both processes.Conclusions Gastrodin exerts anti-fibrotic effects by dually inhibiting the TGF-β/SMAD3 pathway and glycolytic reprogramming, suggesting a novel dual-target strategy for treating liver fibrosis.