A series of plasmid vectors, named pFungiway, was constructed for the purpose of simple and versatile gene manipulation in a wide range of fungi. The backbone of these vectors consists of the binary plasmid pCAMBIA2200, which can be introduced into fungal cells by Agrobacterium tumefaciens-mediated transformation. Objective genes can be easily inserted into the vector by the use of Gateway technology, without relying on any restriction cleavage sites. The pFungiway vectors are composed of two types: expression vectors, in which the expression of the objective genes is driven by a constitutive promoter; and repression vectors, in which the expression of the endogenous target gene is repressed by RNA interference. Either of two genes conferring resistance to hygromycin B or G418 can be used as a selective marker. The availability of pFungiway vectors was confirmed by the use of fluorescent reporter genes in the basidiomycete Flammulina velutipes and in the plant pathogenic fungus Fusarium oxysporum.