Probiotic-derived exopolysaccharides (EPSs) exert significant beneficial effects by regulating the human immune system. In this study, EPS-4412 was extracted from Pediococcus pentosaceus 4412, which was isolated from fermented Manilkara zapota juice. After purification by ion exchange and gel filtration chromatography, its average molecular mass and total carbohydrate content were measured to be 74 kDa and 95.6 ± 0.28 %, respectively. EPS-4412 was characterized as a neutral heteropolysaccharide, primarily composed of mannose with traces of glucose and rhamnose, in a molar ratio of 90.5: 3.48:1, as determined by ultra-high-performance liquid chromatography (UPLC). Fourier transform infrared spectroscopy (FTIR) analysis confirmed the presence of functional groups, OH, CH, CO, and C-O-C in EPS-4412. The existence of α- and β-glycosidic linkages, such as →2)-α-D-Manp-(1→, →3)-α-D-Manp-(1→, →6)-α-D-Manp-(1→, →5)-α-D-Manp-(1→, →4)-α-D-Manp-(1→, →6)-α-D-Glcp-(1 → and →2)-β-D-Rhap-(1 → was elucidated using nuclear magnetic resonance (NMR) spectroscopy. EPS-4412 was further characterized as smooth, glossy, irregular, compact, stacked flaky structures, semi-crystalline, and thermally stable at 252.86 °C using analytical techniques like scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). In vitro anti-inflammatory activity on LPS-stimulated macrophages of EPS-4412 manifested the inhibition of proinflammatory cytokines (IL-6 and TNF-α) and stimulation of the anti-inflammatory cytokine IL-10. Hence, EPS-4412 could be potentially used as a natural additive in the food and pharmaceutical industry.