Abstract: In this study, we investigated the role and potential mechanism of exopolysaccharides (EPS) produced by Limosilactobacillus fermentum in alleviating heavy metal lead toxicity in vitro. A homogenous EPS (B44-EPS) was isolated from L. fermentum B44 in skimmed milk, and its protective effects were evaluated using multiple indicators, including cell viability, intracellular reactive oxygen species (ROS), intracellular lead content, expression of genes encoding mucin, and tight junction proteins in Pb(II)-exposed Caco-2 cells. The B44-EPS weighed 3.05×10⁶ Da) and was composed of glucosamine, glucose, and galactose at a molar ratio of 4.5%:86.6%:8.9%. When Caco-2 cells were co-incubated with 10 mg/L Pb(II) ions for 6 h, B44-EPS at concentrations of 20 μg/mL (low) or 200 μg/mL (high) significantly increased cell viability by 22.08% (P<0.05) and 32.47% (P<0.05), respectively, and reduced intracellular ROS levels by 10.34% (P<0.0001) and 20.19% (P<0.001), respectively, compared to the Pb(II)-exposed control. At the lower concentration of 20 μg/mL, B44-EPS treatment upregulated the mRNA expression of zonula occludens-1 by 96.36% (P<0.01), mucin 2 by 414.29% (P<0.001), and claudin-1 by 89.29% (P<0.01), while downregulating claudin-4 mRNA expression by 95.5% (P<0.0001). Treatment with low and high concentrations of B44-EPS reduced the intracellular lead content by 56.08% (P<0.01) and 80.1% (P<0.001), respectively, compared to the Pb(II)-exposed control. Furthermore, in 10 mg/L Pb(II)-exposed Caco-2 cells, B44-EPS at 20 or 200 μg/mL enhanced transepithelial electrical resistance by 44.52% (P<0.0001) and 43.96% (P<0.0001), respectively, and decreased the permeability of fluorescein isothiocyanate -dextran by 64.91% (P<0.001) and 60.16% (P<0.001), respectively. In conclusion, B44-EPS at 20–200 μg/mL alleviated Pb(II)-induced cytotoxicity in Caco-2 cells by reducing the accumulation of intracellular ROS and Pb(II) and upregulating the expression of genes encoding tight-junction proteins.