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In this study, the yield of exopolysaccharide (EPS) from <i>Lactobacillus plantarum</i> R301 was optimized using a single-factor experiment and response surface methodology (RSM). After optimization, the EPS yield was increased with a fold-change of 0.85. The significant factors affecting EPS production, as determined through a Plackett–Burman design and Central Composite Design (CCD), were MgSO₄ concentration, initial pH, and inoculation size. The maximum yield was 97.85 mg/mL under the condition of 0.01% MgSO₄, an initial pH 7.4, and 6.4% of the inoculation size. In addition, the EPS exhibited strong antioxidant activity, as demonstrated by its ability to scavenge DPPH, ABTS, and hydroxyl radicals. The scavenging rate was up to 100% at concentrations of 4 mg/mL, 1 mg/mL, and 2 mg/mL, respectively. Moreover, the EPS also exhibited reducing power, which was about 30% that of ascorbic acid when both tended to be stable with the increased concentration. These results suggest that <i>L. plantarum</i> R301 EPS possesses different antioxidant mechanisms and warrants further investigation. In addition to its antioxidant activity, the EPS also demonstrated good anti-inflammatory activity by inhibiting the inflammation induced by lipopolysaccharide (LPS) in RAW 264.7 cells, which could decrease nitric oxide (NO) production and expression of the proinflammatory cytokine <i>Il-6</i>. These findings suggest that <i>L. plantarum</i> R301 EPS could be used as a potential multifunctional food additive in the food industry.