Filamentous fungal cells, unlike yeasts, fuse during vegetative growth. The orthologs of mitogen-activated protein (MAP) kinase Fus3 and transcription factor Ste12 are commonly involved in the regulation of cell fusion. However, the specific regulatory mechanisms underlying cell fusion in filamentous fungi have not been revealed. In the present study, we identified the novel protein FsiA as an AoFus3- and AoSte12-interacting protein in the filamentous fungus Aspergillus oryzae. The expression of AonosA and cell fusion-related genes decreased upon fsiA deletion and increased with fsiA overexpression, indicating that FsiA is a positive regulator of cell fusion. In addition, the induction of cell fusion-related genes by fsiA overexpression was also observed in the Aoste12 deletion mutant, indicating that FsiA can induce the cell fusion-related genes in an AoSte12-independent manner. Surprisingly, the fsiA and Aoste12 double deletion mutant exhibited higher cell fusion efficiency and increased mRNA levels of the cell fusion-related genes as compared to the fsiA single deletion mutant, which revealed that AoSte12 represses the cell fusion-related genes in the fsiA deletion mutant. Taken together, our data demonstrate that FsiA activates the cell fusion-related genes by suppressing the negative function of AoSte12 as well as by an AoSte12-independent mechanism.