Bacillus subtilis R 23 produced extracellular biopolymer showing excellent flocculation activity. The biopolymer was confirmed as poly(γ-glutamic acid) (PGA) using high-performance liquid chromatography profile and product characterization. The production, characteristics, and flocculation properties of PGA were studied. PGA produced by B. subtilis R 23 was devoid of any polysaccharides and had a molecular weight of 6.2 × 106 Da. The flocculating activity of PGA in the kaolin suspension was markedly stimulated by the addition of bivalent and trivalent cations in optimum concentration. The pH of reaction mixture also influenced the flocculating activity. Response surface methodology was used to establish the optimum parameters for maximum flocculating activity and to study their interactions. A maximum flocculating activity of 30.32 ± 1.4 1/optical density was obtained with 7.5 mg/L of PGA in combination with 8.0 mM of Ca+2 at pH 7.5.