Heparan sulfate (HS) is a highly sulfated glycosaminoglycan distributed on the cell surface. HS interacts with diverse bioactive molecules called as HS-binding proteins (HSBPs) including growth factors. Although we have revealed the essential roles of HS in various tissues such as pancreatic β-cells and adipose tissue, functions of HS in liver remain largely unknown. In this study, we aimed to elucidate the importance of HS for CCl₄-induced liver injury and regeneration in HepG2 cells, a cell line derived from human hepatocellular carcinoma. Enzymatic removal of HS by heparinase III (HIII) exacerbated CCl₄-induced cytotoxicity, indicating the protective role of HS against liver injury. In order to determine how HS contributes to cell viability, we examined the impact of several HSBPs essential for liver protection and regeneration. Although FGF1 phosphorylated GAB1 and attenuated CCl₄-cytotoxicity through the regulation of CYP2E1 expression in normal HepG2 cells, HIII treatment suppressed the cytoprotective roles of FGF1. We also confirmed the requirement of HS for HGF- and TGFα-induced signaling pathways and cell proliferation after CCl₄ injury. These data demonstrate the essential role of HS in the protection against CCl₄ and the proliferation after injury through the enhancement of growth factor signaling.