The objective of this study was to investigate the dynamic changes in the physicochemical and antioxidant properties of polysaccharides derived from Pediococcus acidilactici (S1-CEPS) through in vitro analysis. A simulated fermentation process resulted in structural modifications of S1-CEPS, accompanied by an increase in its antioxidant activity. Significant enhancements were observed in the content of reducing sugars (p < 0.05), primarily galactose and glucose being released. Moreover, there was a strengthening effect on functional groups such as –OH, –COOH, –CO, and –OSO–O-. Additionally, gut microbes degraded and utilized S1-CEPS to produce short-chain fatty acids (SCFAs), including acetic acid, propionic acid, and n-butyric acid. Furthermore, S1-CEPS exhibited significant modulatory effects on microbial composition by increasing the relative abundances of Proteobacteria and Bacteroidetes while decreasing those of Escherichia-Shigella, Klebsiella, and Fusobacterium. Based on these findings, it is suggested that S1-CEPS derived from Pediococcus acidilactici holds potential as a functional food for promoting health improvement and disease prevention.