Agrobacterium tumefaciensmediated transformation (ATMT)was applied in Penicillium simplicissimum H5 by Agrobacterium tumefaciens LBA4404. The results of hygromycinresistance selection, PCR and Southern blot analysis showed that TDNA was randomly inserted into the transformants with single copy and the transformants indicated genetic stability. The transformation efficiency was about 50 transformants per 105 spores by optimizing the coculture time, the concentration of Agrobacterium tumefacien and acetoyringone (AS).The application of ATMT technique in Penicillium simplicissimum H5 will provide a powerful tool for the genetic engineering of this fungus.