A Ran binding protein (RanBP) homolog, CpRbp1, from Cryphonectria parasitica, has been identified as a protein that is affected by hypovirus infection or tannic acid supplementation. In this study, functional analyses of CpRbp1 were performed by constructing a knockout mutant and analyzing the resulting heterokaryon. Transformation-mediated gene replacement resulted in two putative CpRbp1-null mutants and genotype analyses identified these two mutants as heterokaryotic transformants consisting of two types of nuclei, one with the wild-type CpRbp1 allele and another with the CpRbp1-null mutant allele. Although stable mycelial growth of the heterokaryotic transformant was observed on selective medium containing hygromycin B, neither germination nor growth of the resulting conidia, which were single-cell monokaryotic progeny, was observed on the medium. In trans complementation of heterokaryons using a full-length wild-type allele of the CpRbp1 gene resulted in complemented transformants. These transformants sporulated single-cell monokaryotic conidia that were able to grow on media selective for replacing and/or complementing markers. These results clearly indicate that CpRbp1 is an essential gene, and heterokaryons allowed the fungus to maintain lethal CpRbp1-null mutant nuclei. Moreover, in trans complementation of heterokaryons using chimeric structures of the CpRbp1 gene allowed for analysis of its functional domains, which was previously hampered due to the lethality of the gene. In addition, in trans complementation using heterologous RanBP genes from Aspergillus nidulans was successful, suggesting that the function of RanBP is conserved during evolution. Furthermore, in trans complementation allowed for functional analyses of lethal orthologs. This study demonstrates that our fungal heterokaryon system can be applied effectively to determine whether a gene of interest is essential, perform functional analyses of a lethal gene, and analyze corresponding heterologous genes.