Sulodexide is a heparinoid compound with wide-ranging pharmacological activities. However, the effect of sulodexide on liver fibrogenesis has not been reported. In this study, we aim to evaluate the therapeutic potential of sulodexide in mouse model of liver fibrosis and explore the underlying antifibrotic mechanisms. We found that sulodexide treatment significantly attenuated thioacetamide (TAA) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver fibrosis in mice. Transcriptome analysis revealed that sulodexide treatment downregulated fibrosis-related genes and liver sinusoidal endothelial cells (LSECs) capillarization-associated genes in fibrotic livers. Immunohistochemistry confirmed that the increased expression of LSEC capillarization-related genes (CD34, CD31 and Laminin) in liver fibrotic tissues was reduced by sulodexide treatment. Scanning electron microscopy showed that LSECs fenestrations were preserved upon sulodexide treatment. Quantitative real-time PCR and immunofluorescence demonstrated that the expression of mesenchymal markers was downregulated by sulodexide administration, suggesting sulodexide inhibited endothelial-mesenchymal transition of LSECs during liver fibrosis. Furthermore, sulodexide administration protected primary LSECs from endothelial dysfunction in vitro. In conclusion, sulodexide attenuated liver fibrosis in mice by restoration of differentiated LSECs, indicating that sulodexide treatment may present as a potential therapy for patients with liver fibrosis.