Penicillium expansum causes blue mold in various fruitsproduces patulin, a mycotoxin of significant economicfood safety concern. Fungal infection relies on sensingadapting to environmental cues via G-protein signaling pathways. Despite its importance, the understanding of how these pathways contribute to the infection processpatulin biosynthesis of P. expansum remains limited. Here, we characterized five G protein subunits (PeGαⅠ, PeGαⅡ, PeGαⅢ, PeGβ,PeGγ), all highly expressed during hyphal growthfruit infection. Deletion of these G protein genes leads to various defects in hyphal growth, virulence,patulin production. Notably, ΔPeGαⅠ, ΔPeGβ,ΔPeGγ mutants showed severe defects, with minimal disease symptoms in early infection stagesat least a 50% decrease in patulin production. These mutants displayed straight hyphal growth, reduced branching,significantly decreased vitality. RNA sequencing analysis revealed that this abnormal growth is governed by pathways associated with fungal hyphal polarity. Furthermore, PeGαⅠPeGαⅢ are crucial for cAMP-PKA signaling, while PeGβPeGγ modulate MAPK signaling, both pathways influencing virulencepatulin production. Together, these findings provide evidence that G proteins regulate virulencepatulin production through the regulation of downstream pathwaystheir broader effects on fungal hyphal polarity.