Abstract: Objectives: This study aimed to investigate the preventive and protective effects of gastrodigenin (p-hydroxybenzyl alcohol, HBA) on acute liver injury (ALI) induced by carbon tetrachloride (CCl₄) as well as its regulatory effect on intestinal flora, and to explore its underlying protective mechanism. Methods: A mouse model of ALI induced by CCl₄ was established, and mice were administered different doses of HBA (18, 36, and 72 mg/kg/d) 7 days prior to model induction. The detection indicators included: The serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), the activities of superoxide dismutase (SOD) and catalase (CAT), the liver content of malondialdehyde (MDA), and histopathological examination of liver tissue. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of inflammatory factors, while quantitative polymerase chain reaction (qPCR) and Western Blot were employed to determine the expression levels of inflammatory factors in liver tissue. Additionally, 16S rRNA sequencing technology was applied to analyze the intestinal microbiota composition of in mice based on fecal samples. Results: Compared with the model group, low-, medium-, and high-dose HBA intervention showed protective effedts on CCl₄-induced ALI mice in a dose-dependent manner. High-dose HBA significantly reduced the serum levels of ALT (P<0.01) and AST (P<0.0001), as well as the hepatic content of MDA (P<0.001) in mice; it also increased the activities of SOD (P<0.05) and CAT (P<0.001) and decreased the expression of inflammatory factors in both serum and liver. In addition, HBA effectively alleviated weight loss (P<0.001), liver tissue swelling, and steatosis in mice, and exerted the optimal hepatoprotective effect on ALI mice. Therefore, the high-dose HBA group was selected for 16S rRNA sequencing analysis. Intestinal microbiota analysis revealed that CCl₄ induction altered the species richness of intestinal microbiota; high-dose HBA intervention effectively mitigated the reduction in species richness, improved the microbiota structure at the genus level, promoted the proliferation of the beneficial bacterium Alistipes, and inhibited the colonization of the harmful bacterium Helicobacter. Conclusion: This study confirms that HBA exerts significant protective effects on ALI mice, potentially through enhancing the body’s antioxidant capacity, inhibiting inflammatory responses, and modulating the intestinal microbiota.