Australia has a spectacular biodiversity including animals, plants and fungi. Indigenous Australians have been using higher fungi traditionally as medicines and in religious practice for thousands of years. It has been estimated that there is a large number of different mushroom species present in Australia that are poorly explored and catalogued. Importantly, very little is known about the extent and diversity of lectins from Australian mushroom species [1]. Psathyrella asperospora (Family: Psathyrellaceae) (synonym Lacrymaria asperospora) is an Australian indigenous mushroom from which we have isolated an N-acetyl-D-glucosamine (GlcNAc) specific lectin [1]. De novo sequencing of Psathyrella asperospora lectin (PAL) using LC-MS/MS, identified 10 tryptic peptides that revealed substantial sequence similarity to the GlcNAc specific lectin from Psathyrella velutina (PVL) in both carbohydrate binding and calcium binding sites. Significantly, we also found that PAL has anti-proliferative effect on human colon cancer HT29 cells with an IC50 of 0.48 μM that represents one of the most potent mushroom lectin yet reported [2]. Further characterization of PAL’s anti-proliferative activity using propidium iodine staining revealed that it induced cell cycle arrest at G2/M phase in a manner dependent on its ability to bind GlcNAc on the cell surface. Large scale purification of PAL has now been performed in order to fully characterise the carbohydrate binding specificity including its thermodynamic properties and structural determination using glycan arrays, isothermal calorimetry (ITC) and X-ray crystallography.