Huperzine A (HupA), an anti-Alzheimer’s disease (AD) drug candidate, was isolated from Huperzia serrata. Recently, it was found that various endophytic fungi produce Huperzine A. However, it might be due to the innate properties of endophytic symbiosis, that production of HupA in large quantity from endophytes has not yet been put into practice. Colletotrichum gloeosporioides ES026 was previously isolated from Huperzia serrata and proved to produce HupA. In this study, the strain was successfully transformed using Agrobacterium tumefaciens mediated transformation approach. Moreover, the transformated conditions were optimized. The results showed that under the condition of 25<1×10⁶ spores /mlµA.tumefaciens OD₆₀₀ = 0.3-0.5<400µM acetosyringone and 48h co-cultivation in the presence of induction medium, a high transformation efficiency (about 95 transformants per10⁶ conidia) could be obtained. Under this optimized transformed system, a T-DNA insertional mutant library of 4,000 transformants was constructed. A T-DNA insertional transformant EST010044 was screened, whose yield of Hup A was about 1.83 times of ES026. The border flanking sequence of T-DNA was recovered by TAIL-PCR, and annotated with the highest similarity to a methyltransferase domain. The results provide a foundation for further research of relevant genes of Hup A biosynthesis pathway.