Glutathione peroxidase (GPX) is one of the most important antioxidant enzymes for maintaining reactive oxygen species (ROS) homeostasis. Although studies on fungi have suggested many important physiological functions of GPX, few studies have examined the role of this enzyme in Basidiomycetes, particularly its functions in fruiting body developmental processes. In the present study, GPX-silenced (GPxi) strains were obtained by using RNA interference. The GPxi strains of Hypsizygus marmoreus showed defects in mycelial growth and fruiting body development. In addition, the results indicated essential roles of GPX in controlling ROS homeostasis by regulating intracellular H2O2 levels, maintaining GSH/GSSG balance, and promoting antioxidant enzyme activity. Furthermore, lignocellulose enzyme activity levels were reduced and the mitochondrial phenotype and mitochondrial complex activity levels were changed in the H. marmoreus GPxi strains, possibly in response to impediments to mycelial growth and fruiting body development. These findings indicate that ROS homeostasis has a complex influence on growth, fruiting body development, GSH/GSSG balance, and carbon metabolism in H. marmoreus.