Stichopus japonicus polysaccharides (SJP) were extracted by enzymatic hydrolysis (pepsin and trypsin), polysaccharide content was determined by phenol/sulfate method and the possible mechanism of scavenging reactive oxygen species (ROS) for SJP to protect mitochondria were investigated in this study. The hepatic mitochondrial lipid peroxidation was induced by Fe2+-Vit C system, malondialdehyde (MDA) was used as an index to determine the effect of SJP on lipid peroxidation; effects of SJP on the reducing power and Fe2+ chelating were measured; the scavenging activity of SJP on the hydroxyl radical (·OH) which was produced by H2O2-Fe2+ system was measured; the method of titration and the method of nitrogen blue tetrazolium (NBT) were used to measure the scavenging activity of SJP on the hydrogen peroxide (H2O2) and superoxide anion (O2·−), respectively. The results showed that SJP could inhibit the production of MDA; SJP had a certain reducing power and weaker Fe2+ chelating activity. In addition, SJP could scavenge ?OH, H2O2 and O2·− within a certain concentration range. SJP could protect mitochondria through anti-oxidation and scavenging reactive oxygen species (ROS), which had the effect of protecting the body. This was the possible mechanism of SJP to protect mitochondria.