Antifreeze proteins have shown a great potential in improving the quality of frozen foods, and the isolation of proteins from natural sources that are readily available is deemed to be important. The raw leaf apoplastic extract from cold-acclimated winter wheat grass (Triticum aestivum) containing recrystallization inhibition (RI) proteins was screened for proteolytic and lipolytic activity, and a heat-stable RI protein was isolated. The enzymes detected could be easily removed by heat treatment without any loss in the RI activity. The RI protein was isolated after heat treatment of the raw extract, alcohol precipitation and separation on a size exclusion Chromatographic column. Circular dichroism indicated that the RI protein was mainly β-sheet and random coil, and these structures were preserved up to 75C. After trypsin digestion, mass fingerprinting and sequencing of the digests revealed that the protein belongs to the thaumatin-like protein group.