The psychrotolerant yeast Mrakia frigida 2E00797 isolated from sea sediments in Antarctica was found to be able to produce killer toxin against Metschnikowia bicuspidata, Candida tropicalis and Candida albicans. In the present study, the killer toxin was purified and characterized. The molecular weight of the purified killer toxin was estimated to be 55.6kDa and the purified killer toxin shared 35.1% sequence homology with a protein kinase. The purified killer toxin's optimal temperature and pH for killing activity were 16°C and 4.5, respectively, and it was stable in the temperature range from 10 to 25°C at pH 4.5. The toxin's highest killing activity was observed in the presence of 3.0g/100ml NaCl. The purified killer toxin was able to actively kill whole cells of M. bicuspidata but could not kill the protoplast of the sensitive yeast. Of the eight yeast species tested in this study, the killer toxin was able to kill C. tropicalis and C. albicans in addition to M. bicuspidata.