The antifungal mechanism of dehydrocostus lactone( DHCL) was studied using the target-directed whole cell screen pattern consisting of wild-type Saccharomyces cerevisiae strain WHU2a and three cell wall synthasedeficient strains δfks1,CHS020 and CHS003 derived from WHU2a. The results showed that DHCL could specifically inhibit the growth of glucan synthase-deficient strain δfks1,and the minimum inhibitory concentration( MIC) was 16μg/mL,which was 2 folds lower than that against the wild-type strain and chitin synthase-deficient strains. The growth inhibition on WHU2a could partly be rescued by sorbitol through the growth rescue assay. Furthermore,the content of β-glucan on WHU2a cell wall was decreased by 16. 83% after treated with IC20of DHCL,the activity of β-glucan synthase was inhibited by 26. 66% after treated with 512 μg / mL of DHCL. Based on the data presented,it is reasonable to conclude that the specific inhibition on the β-glucan synthesis in cell wall is one of the main antifungal mechanisms of DHCL.