Targeted gene disruption via biolistic transformation and homologous recombination is a method widely used to identify and investigate the function of genes in Cryptococcus neoformans that causes fatal fungal meningitis if not timely treated. Currently, most laboratories employ the overlap-PCR method to generate a gene-disruption cassette with dominant selectable markers, such as nourseothricin acetyltransferase (NAT). However, the conventional overlap-PCR method is often found to be inefficient because of the presence of multiple templates and of the long length of the final overlap-PCR products. In this report, we suggested an efficient gene-disruption method for C. neoformans, termed a double-joint PCR with NAT-split markers. Here we demonstrated that the gene-disruption cassette generated using double-joint PCR with NAT-split markers can be used successfully for targeted C. neoformans gene disruption with the advantages of providing a more convenient construction of gene-disruption cassettes and high targeted-integration frequency.