In this paper, we reported results about characterization of the factors affecting Agrobacterium tumefaciens-mediated transformation (ATMT) of the plant pathogenic fungus Botrytis cinerea strain RoseBc-3 and about the validity of ATMT for the particular strain of B.cinerea. The results showed that the temperatures for co-incubation of A.tumefaciens and B.cinerea and the conidial concentration of B.cinerea in the co-incubation mixture greatly affected the transformation efficiency. The optimal co-incubation temperature was 22 ℃ and the optimal conidial concentration was 1×105conidia/mL. Under the optimal co-incubation conditions, the numbers of transformant reached 100 per dish (9 cm diameter). All the 16 randomly-selected transformants of B.cinerea were confirmed to harbor the T-DNA of A.tumefaciens based on the result of the specific PCR. The result of the Southern blotting further indicated the integration of the T-DNA in the genome of B.cinerea with the frequency of the single-copy insertion reaching 87.5%. Six types of mutants were identified from the transformants. They were sporulation deficiency mutants (3.8%), mycelial growth-reduced mutants (3.5%), sparse mycelia mutants (1.6%), colony color mutants (5.7%), pathogenicity decreasing mutants (3.3%) and pathogenicity enhancing mutants (3.0%). DNA sequences flanking the T-DNA tag were obtained using the inverse PCR technique and the hi-TAIL PCR technique from 14 mutants of B.cinerea. The insertion sites in these mutants were located with the same frequency in the coding region and in the non-coding regions.