Nef is a lentiviral protein involved in pathogenesis of AIDS, but its molecular mechanism of action remains incompletely understood. Here we report the isolation of the interacting protein with the HIV-1 Nef, using the yeast two hybrid system for expression cloning. One of the positive colonies was selected as the final candidate for the interacting protein gene. The nucleotide sequencing revealed that this interacting protein is Human Ikaros/LyF-1. This protein interacted with the C-terminal region of Nef specifically in yeast system, not with the N-terminal region. This interaction was also confirmed by in vitro binding assay.