Abstract: To investigate purification of ice structuring protein( ISP) from cold- acclimated wheat seedlings,wheat seedlings underwent cold acclimation and ISP from cold- acclimated wheat seedlings was purified by 4 sequential steps including coarse separation,saturated ammonium sulphate precipitation,DEAE- Cellulose 52 chromatography and sephadex- G75 gel filtration chromatography.Differential scanning calorimetry( DSC),heat shock treatments and polyacrylamide gel electro- phoresis( SDSPAGE) were employed respectively for determination of thermal hysteresis activity( THA),ice recrystallization inhibition power( ICP) and molecular weight( MW) of ISPs during process of purification.Survival rate of cold-acclimated wheat seedlings and purification times of ISP during each purification step were calculated as well.Results showed an increase of 240% in survival rate of wheat seedlings using gradient cold- acclimation compared with constant cold- acclimation. Protein content of cold-acclimated wheat seedlings extract( AWSE) was increased by 7.8% in comparison to non- cold- acclimated wheat seedlings extract( NAWSE). Eventual purification times based on THA and ICP were 451.15 and 137.60 respectively. Yield,THA at5 mg / m L,ICP and MW range of ISP were 2.85%,0.180 ℃,70.03% and 20.1 ~31.0 ku respectively. Maximal ICP of ISP was achieved at lower concentration compared with THA.Wheat seedlings can be a potential plant resource in production of ISP.