The exopolysaccharide (EPS) produced by SJC113 is a glucan with α-1,6 and α-3,6 branched glycosidic linkages that may promote human health. The aim of this study was to investigate in vitro the antioxidant, cholesterol-binding, and prebiotic activities of this EPS and its effect on the gut microbiota. The EPS exhibited moderate antioxidant activity, showing free radical scavenging activity (10.94 ± 1.33%) and hydroxyl scavenging activity (6.29 ± 1.59%) at 1 mg/mL. Notably, it showed high cholesterol-binding activity, lowering cholesterol levels by 40% at 1 mg/mL EPS. SJC113 showed strong adhesion to mucin, and its EPS enhanced the adhesion of the probiotic GG. The application of this EPS stimulated the growth of several lactic acid bacteria (LAB) strains in vitro, indicating its potential as a prebiotic. In addition, the use of a human gastrointestinal simulator inoculated with fecal microbiota showed that the EPS favored the growth of spp. and lactobacilli while reducing . These results emphasize the multifunctional nature of the EPS produced by SJC113 with antioxidant, cholesterol-lowering, and prebiotic properties. Further research is required to investigate the specific mechanisms of action and health benefits in vivo.