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Background Liver fibrosis is characterized by endothelial dysfunction and aberrant intercellular crosstalk between endothelial cells and hepatic stellate cells (HSCs); however, the underlying mechanosensitive mechanisms remain incompletely understood. This study aims to investigate the regulatory role of endothelial Piezo1, a mechanosensitive ion channel, in sphingosine-1-phosphate (S1P) synthesis and HSC ferroptosis during liver fibrosis.Methods Single-cell sequencing and metabolomic analyses were performed on liver tissues and serum samples obtained from patients with cirrhosis. Endothelial-specific Piezo1 knockout mice were generated to evaluate fibrotic responses. Mechanistic investigations employed calcium imaging, assessment of endoplasmic reticulum (ER) stress, co-culture systems, chromatin immunoprecipitation, O-GlcNAcylation assays, and a peptide-based targeting strategy.Results Piezo1 expression was markedly upregulated in liver endothelial cells of cirrhotic patients, accompanied by elevated serum S1P levels. Endothelial-specific deletion of Piezo1 significantly attenuated liver fibrosis, as evidenced by reduced collagen deposition and decreased HSC activation. Mechanistically, Piezo1 activation by mechanical stress induced calcium overload and ER stress, promoting endothelial capillarization. Notably, Piezo1 directly interacted with ATF4, facilitating its nuclear translocation and subsequent binding to the Sphk1 promoter, thereby enhancing S1P synthesis in endothelial cells. Endothelium-derived S1P was found to induce FOXK2 O-GlcNAcylation in HSCs, which upregulated SLC7A11 expression and consequently suppressed HSC ferroptosis, a critical protective mechanism against fibrogenesis. Furthermore, a Piezo1-targeting peptide (GsMTx4) conjugated with an endothelial-specific delivery system effectively ameliorated liver fibrosis in vivo, underscoring the translational potential of targeting this pathway.Conclusions Endothelial Piezo1 functions as a critical mechanosensor that drives ATF4-mediated S1P production, which in turn promotes FOXK2 O-GlcNAcylation in HSCs to inhibit ferroptosis and exacerbate liver fibrosis. Targeted inhibition of endothelial Piezo1 represents a promising therapeutic strategy for liver fibrosis.