Rho4　is　a　member　of　the　Rho-family　small　GTPases.　In　this　study,　we　revealed　the　function　of　Rho4　and　explored　its　mechanism　involved　in　intracellular　redox　homeostasis　in　Beauveria　bassiana,　one　of　the　most　widely　utilized　filamentous　entomopathogenic　fungi.　The　disruption　of　Rho4　in　B.　bassiana　resulted　in　significant　phenotypic　changes,　such　as　fungal　virulence,　growth　rate　on　different　media,　thermotolerance,　germination,　and　conidiation.　Integrated　analysis　of　proteomic　and　transcriptomic　data　unveiled　differential　expression　patterns　of　various　redox-related　genes　and　proteins　in　Δrho4,　including　the　down-regulation　of　GST　shown　in　proteomic　and　transcriptomic　data,　and　the　down-regulated　gene　expression　levels　of　NOX,　SOD,　CAT,　and　GR　in　the　transcriptome.　Based　on　the　bi-omics　analysis,　we　focused　on　the　impact　of　Rho4　in　maintaining　intracellular　redox　homeostasis.　A　decreased　ROS　content　observed　in　Δrho4　might　be　attributed　to　the　reduced　NOX　activity,　which　subsequently　affects　the　GSH-producing/consuming　metabolisms,　with　the　attenuated　activities　of　GR　and　GST.　The　imbalanced　redox　homeostasis　also　resulted　in　the　reduced　enzyme　activities　of　SOD　and　CAT.　Exogenous　oxides　could　partially　complement　the　ROS　level　and　rescue　the　growth　defect　in　Δrho4　to　a　certain　extent.　Besides,　BbGDI　was　initially　identified　as　an　interacting　protein　of　Rho4　in　entomopathogenic　fungi.　Our　results　provide　a　comprehensive　understanding　of　the　function　and　regulating　mechanism　of　Rho4　in　B.　bassiana.　©　2024　Elsevier　Inc.