Previous reports have suggested that the Nucleoporin 160 (Nup160) gene of Drosophila simulans (Nup160^sim) causes the hybrid inviability, female sterility, and morphological anomalies that are observed in crosses with D. melanogaster. Here we have confirmed this observation by transposon excision from the P{EP}Nup160^EP372 insertion mutation of D. melanogaster. Null mutations of the Nup160 gene resulted in the three phenotypes caused by Nup160^sim, but revertants of the gene did not. Interestingly, several mutations produced by excision partially complemented hybrid inviability, female sterility, or morphological anomalies. In the future, these mutations will be useful to further our understanding of the developmental mechanisms of reproductive isolation. Based on our analyses with the Nup160^sim introgression line, the lethal phase of hybrid inviability was determined to be during the early pupal stage. Our analysis also suggested that homozygous Nup160^sim in D. melanogaster leads to slow development. Thus, Nup160^sim is involved in multiple aspects of reproductive isolation between these two species.