Abstract: A pullulanase producing strain was isolated from the beach mud of Lianyungang. The strain was identified by morphology, physiological and biochemical analysis and sequencing of 16S rRNA and gyrB genes. The pullulanase was obtained by fermentation and preliminarily purified.The enzymatic properties of the pullulanase were then characterized. Laminaria japonica polysaccharide was hydrolyzed by the crude pullulanase.The antioxidant activity of the hydrolysate of Laminaria japonica polysaccharide was studied. Results showed that the strain P6 was identified as Bacillus megaterium. The specific activity of the pullulanase prepared by fermentation and purification reached 62.3 U/mg. The molecular weight of the enzyme was about 43 kDa. The optimal temperature and pH for the pullulanase activity were 45℃ and 6.5, respectively. After incubation at 40℃ for 5 h, the remaining relative enzymatic activity was above 95%. After incubation at 60℃ for 1 h, the remaining relative enzymatic activity was lost completely. After incubation at pH6~7.5 for 12 h, the remaining relative enzymatic activity was 40%. DPPH and·OH free radical scavenging experiments showed that the antioxidant activity of the polysaccharide was sigificantly enhanced after the hydrolysis of pullulanase (p<0.05). The study provided a new application of pullulanase in food industry.