We have identified an ALG3 homolog (CnALG3), coding for a dolichyl-phosphate-mannose dependent α-1,3-mannosyltransferase, in the human pathogenic yeast Cryptococcus neoformans. The CnALG3 gene encodes a protein of 447 amino acids, showing approximately 33% sequence identity to the homologs of other yeast. For functional analysis of CnALG3, we constructed a null mutant strain and analyzed the N-glycan structures by HPLC and exoglycosidase treatment. CnALG3 was shown to be responsible for the conversion of Man5GlcNAc2-Dol-PP to Man6GlcNAc2-Dol-PP, the earliest step to attach a mannose to the lipid-linked oligosaccharide in the ER. The Cnalg3Δ mutant strain displayed a moderate defect in capsule biosynthesis, and exhibited more increased sensitivity to oxidative and cell wall stresses compared to the wild-type. The C. neoformans phospholipase PLB1, which is a glycoprotein aiding fungal traversal across the blood-brain barrier, was shown to have truncated N-glycans in the alg3Δ, which showed more apparent decrease than in the och1Δ and mnn2Δ. Moreover, the Cnalg3Δ showed fully attenuated virulence in a mouse model of cryptococcosis, suggesting that the alteration of N-glycan assembly affect considerably pathogenicity of C. neoformans. However, the non-opsonic phagocytosis of Cnalg3Δ was shown to be comparable to that of Cncap59Δ during cryptococcal pathogenesis, indicating that the truncated N-linked glycans may not affect the early steps in interaction with macrophages.