Abstract: Pediococcus acidilactici was mutated by space mutagenesis technology, and the strains with high production of exopolysaccharide (EPS) were screened. The functional characteristics of the EPS were also investigated. EPS production and genetic stability of the strains were measured, and a stable mutant strain L21-49 with high production of EPS was selected. Meanwhile, the self-aggregation and hydrophobicity of the mutant strain and the original strain were investigated, and the tolerances of the strains to acid, bile salts, and artificial simulated gastrointestinal fluid were compared. The anti-biofilm, anti-oxidant, and the inhibitory activities on α-amylase and α-glucosidase of EPS were also analyzed. The results showed that the EPS yield of the mutant strain was 196.23 mg/L, which was 26.54% higher than that of the original strain L21. The strain showed good self-aggregation, hydrophobicity and tolerance in vitro. The viable cell number could be maintained at 10⁷ CFU/mL after being cultured in pH2.0 and 3.0 for 3 h. When treated with 4.00 g/L bile salt for 24 h, the number of viable cells were 10⁷ CFU/mL. The viable count could be maintained at 10⁶ CFU/mL after the treatment of the artificial simulated gastrointestinal fluid. The EPS presented good antioxidant capacity in vitro. When the concentration of EPS was 8.0 mg/mL, the clearance rates on DPPH, OH, ABTS⁺ and \rmO_2^- were 91.75%, 38.44%, 54.71% and 58.84%, respectively. The inhibition rates of L21-49 EPS on the biofilms of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Pseudomonas aeruginosa were 40.04%, 60.04%, 50.59% and 33.68%, respectively. It inhibited the activities of α-amylase and α-glucosidase by 23.52% and 51.15%, respectively. In this study, the effects of space mutagenesis on the probiotic characteristics and EPS production of lactic acid bacteria were investigated, so as to provide reference for the research and development of lactic acid bacteria and the EPS.