Background: Ganoderma lucidum Polysaccharides (GLPS) were found to possess various pharmacological properties including anti-inflammatory and hepatoprotective activities. However, the effect and possible mechanism of GLPS treatment on liver injury have not yet been reported. Therefore, this study aimed to explore the potential anti-inflammatory and hepatoprotective effects and possible mechanism of GLPS in carbon tetrachloride (CCl4)-induced acute liver injury mice. Summary: GLPS significantly reduced the activation of NLRP3 inflammasome and improved liver function in liver injury mice. It significantly inhibited CCl4-induced changes of alanine aminotransferase and aspartate aminotransferase activities in serum, as well as nitric oxide synthase (NOS) and cytochrome P450 2E1 (CYP2E1) activities in liver tissue; it also remarkably decreased levels of liver weight and index, total bilirubin, interleukin (IL)-1β, IL-18, IL-6 and tumor necrosis factor-α in serum, as well as malondialdehyde and IL-1β in liver tissue. Protein expression levels of liver NLRP3, ASC, and Caspase-1 were also downregulated, while the glutathione level in liver tissue was remarkably enhanced in GLPS groups compared to that of the model group. Key Message: These results suggested that GLPS may be a potential for the prevention and treatment of acute liver injury with liver inflammation. The possible mechanism may be related to the inhibition of free radical lipid peroxidation, NOS, and CYP2E1 activities and activation of liver inflammatory factors.