Abstract: To study the antagonism of Saccharomyces Cerevisiae's extracellular proteins against Kluyveromyces Thermotolerans in the mixed culture, and to preliminarily separate the toxic proteins, extracellular proteins of Saccharomyces Cerevisiae from single culture and dialysis tube fermentation were extracted by ultrafiltration, added into the single culture of Kluyveromyces Thermotolerans respectively, for further population dynamic analysis of Kluyveromyces Thermotolerans under the influence of Saccharomyces Cerevisiae extracellular proteins by plate count method.The extracellular proteins of Saccharomyces Cerevisiae from the two cultures were seperated and analyzed using two-dimensional electrophoresis combined with PDQuest software.Statistics showed that extracellular proteins of Saccharomyces Cerevisiae from single culture displayed no antagonistic action on Kluyveromyces Thermotolerans, while those extracted from the dialysis tube fermentation induced the early death Kluyveromyces Thermotolerans.79 spots were detected on the single culture profile while 109 spots were found on the dialysis tube fermentation one.By comparison, 30 new spots appeared on the dialysis tube fermentation image.In conclusion Saccharomyces Cerevisiae could secrete high molecular weight (>10ku) proteins that could be toxic to Kluyveromyces Thermotolerans.Moreover, the secretion of toxic proteins was not spontaneous, but by the induction of secretion smaller than 10ku from non-Saccharomyces Cerevisiae.The antagonistic proteins were included within the 30 new spots.