Fusarium graminearum is an economically devastating pathogen that causes cereal worldwide. The plant disease cycle involves sexual reproduction, with the perithecium playing a crucial role in overwintering and the discharge of ascospores. Although fatty acid biosynthesis and metabolism are linked to perithecium formation and ascospore discharge, the regulation of these processes remains largely unknown. In this study, we identified and characterized FgBZIP16, as a basic leucine zipper (b-ZIP) transcription factor, in F. graminearum. Targeted gene deletion revealed that FgBzip16 is important for vegetative growth, asexual/sexual development, and plant infection. Cytological observations revealed that FgBzip16 was localized in the nucleus during the hyphal and conidial stages. FgBzip16 is essential for ascospore discharge, with transcriptomics and molecular biology showing it binds to the promoter of its target genes FGSG_05321 and FGSG_03244, which regulate ascospore discharge by encoding fatty acid synthase subunit alpha-reductase and enoyl hydratase, respectively. Altogether, these results constitute the first report of the specific functions associated with b-ZIP transcription factor FgBzip16, linking its regulatory roles to fungal development, fatty acid accumulation, and metabolism, host penetration, and pathogenicity of F. graminearum.