alpha-Glucosidase I regulates trimming of the terminal alpha-1,2-glucose residue in the N-glycan-processing pathway, which plays an important role in the quality control system in mammalian cells. However, the consequence of glucose trimming of the N-glycan in filamentous fungi is unclear. We identified the gene encoding alpha-glucosidase I in the human opportunistic fungal pathogen Aspergillus fumigatus, namely Afcwh41. Deletion of the Afcwh41 gene resulted in a defective N-glycan processing of the proteins secreted by A. fumigatus. Although the Afcwh41 was not essential for hyphal growth and virulence, a severe reduction in conidia formation and a temperature-sensitive deficiency of cell wall integrity (CWI) were observed. Also, abnormalities of polar growth and septation were observed during conidial germination and hyphal elongation of the mutant. Our results suggest that Afcwh41 was involved in CWI, polarity, septation, and conidiation in A. fumigatus, probably by affecting the proper function of the proteins that are required for cell wall synthesis.