A Cryptococcus neoformans var. grubii strain, NUBS14020, was the first fluconazole (FLZ)-resistant strain isolated from a feline cryptococcosis. Subsequent work demonstrated that multi-azole-resistant strains are readily isolated from FLZ-resistant strains by culturing in medium containing voriconazole (VRZ). The resulting clones were assessed for mutation and expression of known target genes, including the loci encoding lanosterol 14-alpha demethylase (ERG11), an ATP-binding cassette (ABC) transporter (AFR1), or a multidrug efflux pump (MEP); mutation and/or overexpression of these genes is known to be associated with azole resistance. We also examined the interaction between an efflux blocker (FK506, calcineurin inhibitor) and VRZ in themulti-azole-resistant strains. The ERG11 genes frommulti-azole-resistant strains encoded a protein with a G344S substitution. Expression levels of AFR1, ERG11, and MEP in the multi-azole-resistant strains were not higher than those in the VRZ-susceptible parent strain (NUBS14020) when cultured in Sabourad's dextrose broth containing VRZ. Synergistic effects between FK506 and VRC were observed in all of the multi-azole-resistant strains. The minimal inhibitory concentrations (MICs) of the combination of VRZ and FK506 in multi-azole-resistant strains were 4 to 8 times lower that the MICs of VRZ alone. To the best of our knowledge, this work represents the first report thatmulti-azole-resistant strains of C. neoformans encode a G344S substitution in Erg11p. Further investigation will be needed to determine the mechanism of multi-azole resistance in C. neoformans, given that feline cryptococcosis due to multi-azole-resistant strains is readily transmitted from cats to humans.