Saccharomyces boulardii is the best knownprobiotic yeast, widely used as a therapeutic agent for thetreatment or prevention of diarrhea and intestine disorders. In the present work, we established a target gene disruptionsystem for S. boulardii based on the Cre-loxP system usedfor S. cerevisiae and other fungi by screening out selectionmarkers, working out the transformation method, andconstructing essential plasmids for S. boulardii. Theestablished system was successfully applied to the URA3gene disruption and created an ura3 null mutant strain ofS. boulardii. The system can be used for PCR mediatedgene disruption, cloning mediated gene disruption, andreintroduction of the deleted gene back to the mutant. Allthe introduced exogenous DNAs in the gene disruptionprocedures were removed from the final mutant strainexcept the two 34 bp loxP pieces left in deleted gene loci.