This work aimed to investigate the in-vitro and in-vivo fermentation behaviors of Holothuria leucospilota Polysaccharides (HLP) and the impact on mouse liver antioxidant activity. HLP showed excellent fermentability during in vitro experiments, which was characterized by increased levels of total sugar consumption and short-chain fatty acids (SCFAs). During in vitro fecal fermentation, the fucose contents in the HLP fermentation products (0.174 mg/mL) were higher than those of xylose and galactosamine during the first three hours, and fucose disappeared after 24 h. The concentrations of the generated SCFAs increased to 111.13 mmol/mL after in-vitro fermentation at 48 h. After 28 days of oral administration, the SCFA contents that were detected in the feces of mice treated with high HLP doses were significantly higher than those in the feces of mice treated with lower doses and the normal group. In addition, histological observations demonstrated that HLP increased the number of goblet cells without causing hepatocellular injury. Moreover, the increased glutathione peroxidase (GSH-Px) and superoxidase dismutase (SOD) activities and decreased malondialdehyde (MDA) contents in the mouse livers treated with HLP suggested the good performance of HLP with respect to liver antioxidants.