Colletotrichum acutatum was the main cause of the recent outbreaks of anthracnose on pepper fruit in Korea. To facilitate molecular analysis of C. acutatum, we generated an arginine auxotrophic mutant of the C. acutatum strain JC24 using a targeted gene replacement strategy. A 3.3-kb genomic region carrying an ortholog (designated CaARG2) of the fungal gene encoding Nacetylglutamate synthase, the first enzyme of arginine biosynthesis in fungi, was deleted from the fungal genome. The mutant exhibited normal growth only when arginine was exogenously supplied into the culture medium. Transformation of the arginine auxotrophic mutant with a plasmid DNA carrying an intact copy of CaARG2, which was smaller than the deleted region in the mutant, not only caused random vector insertions in the fungal genome, but also recovered both hyphal growth and pathogenicity of the mutant to the wild-type level. Using this new selection system, we have successfully developed a restriction enzyme-mediated integration procedure, which would provide an economically efficient random mutagenesis method in C. acutatum.