Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation (ATMT), but the frequencies are often very low. In the present study, we replaced the Ku80 homolog (a gene of the non-homologous end-joining (NHEJ) pathway) with the hygromycin resistance cassette (hph) by ATMT. No significant change in vegetative growth, conidiation, or pathogenicity was observed in Ku80-deficient strain (ΔPdKu80) of P. digitatum. However, using ΔPdKu80 as a targeting strain, the gene-targeting frequencies for both genes PdbrlA and PdmpkA were significantly increased. These results suggest that Ku80 plays an important role in homologous integration and the created ΔPdKu80 strain would be a good candidate for rapid gene function analysis in P. digitatum.