Abstract: The antagonistic yeast N1 with a high ability of inhibiting the gray mould (Botrytis cinerea) disease on strawberry was isolated from the strawberry fruits and leaves in Nanjing Maqun with in vitro and in vivo methods. This strain was preliminarily identified as Pichia guilliermondii based on the similarity analysis of its physiological-biochemical characteristics and 26S rDNA D1/D2 domain sequence. Pretreatment with different concentrations of N1 could decrease the incidence of rot on postharvest strawberry after inoculation with pathogenic fungi Botrytis cinerea. The result showed that the higher concentration of N1, the inhibition effect on the gray mould disease was better. The best inhibition concentration of N1 was 5×108CFU/mL which could decrease the incidence of rot to 32. 3%, compared with rot only inoculating with pathogenic fungi. Postharvest strawberries treated with the antagonist were stored at (2±1) ℃ with RH 90%～95%, the results showed that the antagonist treatment had little effect on acid content, but could maintain fruit luster, firmness, soluble solid and vitamin C content, also reduce weight loss rate remarkably and extend storage life. These results revealed a potential commercial application of N1 as an antagonist for controlling the fruit postharvest diseases.