The nsdD gene, encoding an unique GATA factor NsdD, plays a pivotal role in sexual develoment not only in Aspergillus nidulans as well as many other filamentous fungi. The Ind proteins in A. nidulans were identified as interactors of NsdD protein by using GST-pull down assay and co-immunoprecipitation. IndB and IndD were confirmed to be bound to the zinc finger domain of NsdD through mapping of the NsdD interaction domains. Interestingly, IndB or IndD proteins do not show functional homogeneity with domain of any protein characterized to date. A single mutation in indB or indD was normal in sexual development, but double mutations in indB and indD resulted in uncontrolled formation of mature and immature cleiostothecium during sexual development, suggesting that Ind proteins play as negative regulators of sexual development in A. nidulans. Based on these results, we analyzed the intracellular localization of the interaction between NsdD and Ind proteins in A. nidulans using the split-YFP/BiFC method. YFP signals of NsdD-IndB, NsdD-IndD and IndB-IndD were observed in the vesicle domes and metulae of the conidiophore, an asexual reproductive organ, and fluorescence was also observed in the septum and cell wall of hyphae.