The "p53-like" superfamily transcription factor, VIB-1, plays a crucial role in mediating heterokaryon incompatibility and regulating the transcription of specific genes involved in the secretion of extracellular hydrolases in Neurospora crassa. However, the precise mechanism underlying the transcriptional regulatory function of VIB-1 is still poorly understood. Here, we reveal that VIB-1 is involved in the H2O2-induced oxidative stress response, in which deletion of vib-1 leads to an H2O2-sensitive phenotype and inhibition of cat-3 expression. Conversely, VIB-1 overexpression confers an H2O2-resistant phenotype and robustly activates cat-3 in a dose-dependent manner. Importantly, we identified the DNA-binding domain of VIB-1 as the key component required for these regulatory processes. Furthermore, VIB-1 activates cat-3 transcription by interacting with and recruiting general transcription factors and RNA polymerase II to the cat-3 promoter, resulting in eviction of H2A.Z and a decrease in nucleosome density in these regions. Additionally, VIB-1 positively regulated the expression of other two target genes, NCU05841 and NCU02904, in the same manner. Together, our findings reveal a mechanism by which VIB-1 is involved in the transcriptional activation of cat-3 and other VIB-1-targeted genes by promoting PIC assembly on their promoters.