Fusarium graminearum, the causal agent of Fusarium head blight in cereal crops, produces sexual progeny (ascospore) as an important overwintering and dissemination strategy for completing the disease cycle. This homothallic ascomycetous species does not require a partner for sexual mating; instead, it carries two opposite mating-type (MAT) loci in a single nucleus to control sexual development. To gain a comprehensive understanding of the regulation of sexual development in F. graminearum, we used in-depth and high-throughput analyses to examine the target genes controlled transcriptionally by two-linked MAT loci (MAT1-1, MAT1-2). We hybridized a genome-wide microarray with total RNAs from F. graminearum mutants that lacked each MAT locus individually or together, and overexpressed MAT1-2-1, as well as their wild-type progenitor, at an early stage of sexual development. A comparison of the gene expression levels revealed a total of 1,245 differentially expressed genes (DEGs) among all of the mutants examined. Among these, genes involved in metabolism, cell wall organization, cellular response to stimuli, cell adhesion, fertilization, development, chromatin silencing, and signal transduction, were significantly enriched. Protein binding microarray analysis revealed the presence of putative core DNA binding sequences (ATTAAT or ATTGTT) for the HMG (high mobility group)-box motif in the MAT1-2-1 protein. Targeted deletion of 106 DEGs revealed 25 genes that were specifically required for sexual development, most of which were regulated transcriptionally by both the MAT1-1 and MAT1-2 loci. Taken together with the expression patterns of key target genes, we propose a regulatory pathway for MAT-mediated sexual development, in which both MAT loci may be activated by several environmental cues via chromatin remodeling and/or signaling pathways, and then control the expression of at least 1,245 target genes during sexual development via regulatory cascades and/or networks involving several downstream transcription factors and a putative RNA interference pathway.