Hirsutella satumaensis, an endoparasitic fungus that targets Lepidoptera insects, holds significant potential for biocontrol applications. However, its molecular study has been limited due to the absence of an efficient genetic transformation system. In this study, an optimized Agrobacterium tumefaciens-mediated transformation protocol was developed for H. satumaensis using binary vectors pBARGPE1-GFP and pK2-bar, which carry the green fluorescent protein (eGFP) and phosphinothricin resistance (bar) genes, respectively. The optimal transformation conditions included a conidial concentration of 10⁵ conidia/mL, an A. tumefaciens (strain AGL-1) concentration of OD