Abstract: In order to increase the microbial flocculating polysaccharide production by Paenibacillus sp. A9, the response surface methodology (RSM) was used to analyze the fermentation conditions. The P-B (Plackett-Burman) results showed that the amount of media, ρ(MgSO₄·7H₂O) and ρ(soluble starch), were three main factors. The culture medium and conditions were optimized according to the Box-Behnken design and RSM analysis. Moreover, a maximum flocculating polysaccharide production of 2.49g/L was obtained with the optimal conditions of 17g/L ρ(soluble starch), 3.0g/L ρ(yeast extract), 6g/L ρ(K₂HPO₄), 0.2g/L ρ(MgSO₄·7H₂O), 0.10g/L ρ(NaCl), 51mL of media amount and pH 8; the value was very close to the predicted value of 2.50g/L. The infrared spectra showed that the flocculating polysaccharide produced by strain A9mainly contains polar groups, such as hydroxyl (—OH), carboxyl (—COO^-), methoxyl (—C—O—C—) and acetyl amino group (—NHCOCH₃). Combined with the experimental results of different carbon sources utilization, it is speculated that the main ingredients of the microbial flocculant produced by strain A9are polysaccharides such as α-D-mannopyranose, α-D-glucopyranose, acidic polysaccharides and acetyl amino polysaccharide.