Abstract: Gardenia jasminoides Ellis can provide two functions: medicine and food, due to various active ingredients, such as geniposide, gardenia yellow pigment, polysaccharides, organic acids, flavonoids, and volatile oil. The content of polysaccharides in Gardenia can range from 4% to 11%, leading to the primary efficacy of Gardenia system. Current research of Gardenia has focused mainly on the geniposide and gardenia yellow pigment. It is very limited on the polysaccharide in recent years. This study aims to fully utilize the polysaccharide components in Gardenia. A series of methods were also established to optimize the extraction, separation and purification for Gardenia polysaccharides. A systematic investigation was then implemented to explore the antioxidant activity of the polysaccharides in vitro. Among them, crude polysaccharides were extracted from Gardenia using ultrasonic-assisted method. Single-factor and orthogonal tests were carried out to optimize the purification of the crude polysaccharides, according to anion exchange resin. Specifically, the antioxidant activity of the purer polysaccharide was firstly evaluated using reduced power and free radical scavenging assay. An analysis was made to determine the reducing power of the pure polysaccharide and its scavenging rates of hydroxyl radical (OH), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), and diammonium 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) radical (ABTS^+·). The vitamin C (VC) was taken as the control. The leghorn male hepatoma (LMH) chicken hepatocytes were cultured in vitro, in order to investigate the antioxidant of the pure polysaccharide at the cellular level. The cytotoxicity of the polysaccharide towards LMH cells was also assessed using cell viability assays. Subsequently, the LMH cells were subjected to a 1-hour pretreatment at 42°C. A heat-stressed cell model was established to induce the oxidative damage. The different concentrations of the polysaccharides were utilized after purification for a specific duration. The markers of oxidative damage were then detected to define the specific level in the LMH cells. The optimal combination of extraction parameters was determined as follows. A solid-liquid ratio of 1:20; an extraction time of 50 min; a temperature of 80 ℃; and one extraction cycle. The yield of crude polysaccharides from Gardenia was 11.5% under the optimal condition. The purified polysaccharide, named GP, was obtained after deproteinization, decolorization, and desalting. The polysaccharide content of GP was determined to be 45.3% (expressed in glucose) by phenol-sulfuric acid method. It was found that the reducing power of GP and the scavenging rates of·OH, DPPH·and ABTS+·significantly rose with the increase in GP concentration The maximum reducing power of GP was approximately half of that of 0.5 mg/mL VC, and the maximum scavenging rates of all free radicals were similar to those of 0.5 mg/mL VC. The median effective concentrations (EC₅₀) of GP for the scavenging OH, DPPH and ABTS+ were 3.8, 0.1 and 1.1 mg/mL, respectively. There was no influence on the viability of LMH cells, in terms of the GP concentrations ranging from 0.1 to 0.4 mg/mL (P＞0.05). The levels of reactive oxygen species (ROS), aspartate aminotransferase (AST), and malondialdehyde (MDA) were significantly reduced in the GP group (P＜0.05), compared with the heat stress group. Meanwhile, there was the significant increase in the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the total antioxidant capacity (T-AOC) (P＜0.05). The better performance was achieved in the parameters comparable to the positive and the blank control group. In summary, the polysaccharides after optimization shared the strong in vitro antioxidant activity even to effectively scavenge the free radicals. Furthermore, the polysaccharides were alleviated the oxidative damage to chicken hepatocytes that induced by heat stress. As such, the activity of antioxidant enzymes was enhanced to reduce the oxidative damage markers in the damaged cells. Therefore, the Gardenia polysaccharides can be expected to serve as the effective antioxidant in feed additives industries.