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