Microtubules composed of α- and β-tubulin dimers are major components of the cytoskeleton. They are involved in mechanical support, cytoplasmic organization as well as in a number of cellular processes, such as chromosome separation, cell migration and organelle transport. The role of microtubules in those processes are related to diverse microtubules-associated proteins, such as plus-end tracking proteins for dynamics of microtubules, motor proteins for vesicle trafficking, and tubulin-folding cofactors for microtubules organization. A common feature of these proteins is the presence of a cytoskeleton-associated protein-glycine-rich (CAP-Gly) domain. CAP-Gly domain is evolutionarily conserved and generally considered to bind to α-tubulin to regulate functions of microtubules. However, there has been a dearth of research on CAP-Gly proteins in human fungal pathogen Cryptococcus neoformans, which causes fatal meningoencephalitis predominantly in immunocompromised patients. In this study, we identified three CAP-Gly protein encoding genes (CNAG_06352, CNAG_03702 and CNAG_01051) by performing blast analysis of CAP-Gly domain amino acid sequence on the C. neoformans genome database. We successfully constructed deletion mutants of alf1Δ (CNAG_03712) and cgp1Δ (CNAG_06352) in serotype A MATα H99. ALF1, which is a homolog of the Saccharomyces cerevisiae ALF1, plays a similar function to mammalian tubulin cofactor B. CGP1 encodes a specific CAP-Gly protein with a unique domain structure and thereby named Cgp1 (CAP-Gly protein 1). When alf1Δ mutant was exposed to microtubule destabilizer-thiabendazole (TBZ), no distinguishable phenotype was observed. On the other hand, both cgp1Δ mutant and the overexpression strain of CGP1 exhibited significant growth defect in response to TBZ. We also observed co-localization of Cgp1 with cytoplasmic microtubules and verified the binding between Cgp1 and α- tubulin by in vivo pull down assay, which further confirmed that Cgp1 is involved in the stability of microtubules in C. neoformans. In addition, we identified the role of Cgp1 in maintenance of membrane stability and genotoxic stress responses by observing sensitive phenotypes of cgp1Δ mutant. Furthermore, in order to characterize the function of Cgp1 in differentiation, cgp1Δ mutant in MATa KN99 was constructed and crossed with cgp1Δ mutant in MATα H99. We discovered a rather surprising role of Cgp1 in regulating differentiation during developmental stages as the deletion of CGP1 promoted filament formation and led to a severe defect in cell fusion. Moreover, cgp1Δ mutant showed attenuated virulence in insect model (Galleria mellonella) indicating that Cgp1 is involved in virulence of C. neoformans. Finally, we constructed domain deletion strains, in which each domain was selectively deleted, to characterize the role of each domain in Cgp1. The results demonstrated that the CAP-Gly domain plays a crucial role in Cgp1 among the Cgp1 containing domains (SPEC and Phage-GP20). In conclusion, Cgp1 as a CAP-Gly protein has conserved and distinct roles in regulating growth and pathogenicity of C. neoformans.