Soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) play a crucial role in the development and virulence through mediation of membrane fusion and vesicle trafficking in pathogens. Our previous studies reported that the SNARE protein FgVam7 and its binding proteins FgVps39/41 are involved in vesicle trafficking and are important for vegetative growth, asexual/sexual development, deoxynivalenol production and virulence in the Fusarium head blight fungus Fusarium graminearum. Here, we identified and characterized another FgVam7 binding protein in F. graminearum, FgPep12, an ortholog of yeast t-SNARE Pep12 with both the SNARE and TM domains being essential for its localization and function. Deletion of FgPep12 caused defects in vegetative growth, conidiogenesis, deoxynivalenol production and virulence. Cytological observation revealed that FgPep12 localizes to the Golgi apparatus, late endosomes and vacuoles, and is necessary for transport from the vacuole to prevacuolar compartment. Further investigation revealed that both FgPep12 and FgVam7 are essential for ascospore discharge through interaction with and trafficking of the Ca2+ ATPase FgNeo1 between the Golgi and endosomal/vacuolar system. FgNeo1 has similar biological roles to FgPep12 and is required for ascospore discharge in F. graminearum. Together, these results provide solid evidence to help unravel the mechanisms underlying the manipulation of ascospore discharge and plant infection by SNARE proteins in F. graminearum.