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Agrobacterium tumefaciens-mediated DNA transformation method was applied to transform Nodulisporium sylviforme fusant HDF-68, a taxol-producing fungus. We constructed a binary vector pBI121-43 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of N.sylviforme with A.tumefaciens containing pBI121-43 led to 110～130 hygromycin-resistant transformants per million conidia. Putative transformants were found to be mitotically stable. The molecular analysis of transformants demonstrated the random integration of single copy of the T-DNA into the host genome. This transformation system serves as a basic tool for insertional mutagenesis in N.sylviforme fusant HDF-68, and the development of such system lays a solid foundation for constructing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.