Exopolysaccharides (EPSs) have been shown to possess wide-ranging of physiological functions, including antioxidant, anticancer, and immunomodulatory. However, there is a scarcity of research focused on the probiotic function of EPSs isolated from Bifidobacteria, and further investigation is needed to reveal the mechanisms underlying their functions. In this study, EPSs were isolated and purified from Bifidobacterium animalis subsp. Lactis SF (SF-EPS), and the probiotic potential of SF-EPS were analyzed by in vitro antioxidant capacity assays and human fecal fermentation. Followed by the evaluation of the eps cluster in B. lactis SF. The results showed that SF-EPS (MW: 4.81 x 104 Da) contained glucose, galactose, arabinose, and mannose in a molar ratio of 4.02:1.91:1.02:1.0 and small amounts of glucuronic acid (0.02 +/- 0.01 mol%). Furthermore, SF-EPS showed strong antioxidant capacity, which was embodied in its ability of scavenging DPPH and hydroxyl radicals. SF-EPS regulated the gut microbiota by increasing the relative abundances of Faecalibacterium, Anaerostipes, and Bifidobacterium and decreasing the abundance of Enterobacter and Klebsiella. SF-EPS promoted the production of short-chain fatty acids (SCFAs) by intestinal microorganisms. Therefore, SF-EPS has the potential for utilization as a prebiotic in functional foods, enhancing gut health, and offering a natural ingredient for use in industrial food production.