The S-type lectins in annelida are different in molecular structure and biochemical properties from that of common galectin and are valuable in anticancer study. Based on their specificity, Sepharose CL-6B affinity chromatography was adopted in this study to separate the S-type lectin in annelida from earthworms, in which EDTA-MEPBS (2 mmol/L EDTA, 4 mmol/L beta-mercaptoethanol, 150 mmol/L NaCl, 20 mmol/L phosphate, pH 7.2) was used as an equilibrium solution and aqueous ammonia solution (150 mmol/L, pH 10.5) as an eluent. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the relative molecular mass of the obtained protein in this purification procedure was about 32 000 and it could be further separated into fractions with relative molecular mass about 15 000. Hemagglutination experiment and fluorescence analysis demonstrated that this protein possessed the characteristics of agglutinin and its complex with lactose. This indicated that the target protein was one of the S-type lectins in annelida. This study may offer a novel and rapid method for purifying the S-type lectins in annelida in large scale.