Abstract: Polypeptide fermented from konjac powder was separated into three segments with different molecular weight:M>200 ku,10 ku<M<200 ku,M<10 ku by ultrafiltration. Sephadex G-100 gel chromatography was used to ulteriorly separate the polypeptide segment of 10 ku <M<200 ku,three components of N1,N2 and N3 were obtained respectively. After the optimization on elution conditions,the optimal conditions for Sephadex G-100 separation was confirmed as follows:150 mg/m L of sample concentration,0.8 m L/min of elution flow rate. The HPLC showed that the amino acids profile of N2 had high similarity to the standard mannatide and the mannatide was up to 56.78%±2.41%,which suggested the mannatide from polypeptide mainly distributed in N2 component.