The Woronin body, a unique organelle found in the Pezizomycotina, plugs the septal pore upon hyphal damage to prevent excessive cytoplasmic bleeding. Although it was previously shown that the Woronin body buds out from the peroxisome, the relationship between peroxisomal proliferation/division and Woronin body differentiation has not been extensively investigated. In this report, we examined whether Pex11 required for peroxisomal proliferation participates in Woronin body formation in Aspergillus oryzae. A. oryzae contained two orthologous PEX11 genes that were designated Aopex11-1 and Aopex11-2. Deletion of Aopex11 genes revealed that only the Delta Aopex11-1 strain showed reduced growth and enlarged peroxisomes in the presence of oleic acid as a sole carbon source, indicating a defect in peroxisomal function and proliferation. Disruption of Aopex11-1 gene impaired the Woronin body function, leading to excessive loss of the cytosol upon hyphal injury. Dual localization analysis of the peroxisome and Woronin body protein AoHex1 demonstrated that Woronin bodies fail to fully differentiate from peroxisomes in the Delta Aopex11-1 strain. Furthermore, distribution of AoHex1 was found to be peripheral in the enlarged peroxisome or junctional in dumbbell-shaped peroxisomes. Electron microscopy of the Delta Aopex11-1 strain revealed the presence of Woronin bodies that remained associated with organelles resembling peroxisomes, which was supported from the sucrose gradient centrifugation confirming that the Woronin body protein AoHex1 overlapped with the density-shifted peroxisome in the Delta Aopex11-1 strain. In conclusion, the present study describes the role of Pex11 in Woronin body differentiation for the first time.