The antifungal peptides, drosomycin (Drs) and its isoform drosomycin-like C (Drs-lC) from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-lC, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-lC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-lC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains. The antifungal peptides, drosomycin (Drs) and its isoform drosomycin-like C (Drs-lC) from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with filaments utilizes the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-1C, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-lC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the product s of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-lC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test