By using Agrobacterium tumefaciens-mediated transformation, we successfully transformed Fusarium verticillioides and obtained T-DNA insertion mutants. Under the optimal condition of 106spores/mL, A. tumefaciens OD600=0.15-0.20, 200μg/mL acetosyringone and 36h co-cultivation, the transformation efficiency reached 60-120 transformants per 106spores. More than 1000 transformants were obtained. Most of them were quite stable after five rounds of successive cultures. PCR amplification showed that the T-DNA was integrated into the genome, and was stable through mitotic cell division. The transformation system is the basis for study of pathogenicity mechanism and functional gene of the fungi.