The nucleoporin Nup98 is an essential component of the nuclear pore complex. This peripheral nucleoporin with its Gly-Leu-Phe-Gly (GLFG) repeat domain contributes to nuclear-cytoplasmic trafficking, including mRNA export. In addition, accumulating studies indicate that Nup98 plays roles in several important biological events such as gene expression, mitotic checkpoint, and pathogenesis. Nup98 is well conserved among organisms belonging to the fungi and animal kingdoms. These kingdoms belong to the eukaryotic supergroup Opisthokonta. However, there is considerable diversity in the Nup98 orthologs expressed in organisms belonging to other eukaryotic supergroups. Intriguingly, in ciliates, a unicellular organism having two functionally distinct nuclei, GLFG-Nup98 is present in one of the nuclei and a distinct Nup98 ortholog is present in the other nucleus, and these different Nup98s participate in a nucleus-selective transport mechanism. In this review, we focus on Nup98 function and discuss how this nucleoporin has evolved in eukaryotic kingdoms.