Cryptococcus gattii R265 is a hyper-virulent fungal strain responsible for the major outbreak of cryptococcosis in Vancouver Island of British Columbia in 1999. It differs significantly from C. neoformans in its natural environment, its preferred site in the mammalian host, and in the nature and mode of pathogenesis. Our previous studies in C. neoformans have shown that the presence of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in the host, while its absence induces robust immune responses, which in turn facilitate clearance of the fungus and induces a protective response. The results of the present investigation reveal that the cell wall of C. gattii R265 contains 2-3-fold higher amount of chitosan compared to that of C. neoformans. The genes responsible for the biosynthesis of chitosan are highly conserved in the R265 genome; the roles of the three chitin deacetylases (CDA) have however, been modified. To deduce their roles, single, double and a triple CDA deletion strains were constructed in a R265 background and were subjected to mammalian infection studies. Unlike C. neoformans where Cda1 has a discernible role in fungal pathogenesis, in R265 Cda3 is critical for virulence. Deletion of either CDA3 alone (cda3Δ) or in combination with either CDA1 (cda1Δ3Δ) or CDA2 (cda2Δ3Δ) or both (cda1Δ2Δ3Δ) rendered the yeast cells avirulent and were cleared from the infected host. Moreover, the cda1Δ2Δ3Δ strain of R265 induced a protective response to a subsequent infection with R265. These studies shed more light into the regulation of chitosan biosynthesis of C. gattii and its subsequent effect on fungal virulence.