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Phagocytic amoeboid predators such as amoeba have been proposed to select for survival traits in soil microbes such asCryptococcus neoformansthat can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Several prior studies have shown that incubation of various fungal species with amoeba can enhance their virulence. However, the mechanisms by which fungi adapt to amoeba and thus change their virulence are unknown. In this study we exposed three strains ofC. neoformans(1 clinical and 2 environmental) to predation byAcanthamoeba castellaniifor prolonged periods of time and then analyzed surviving colonies phenotypically and genetically. Surviving colonies were comprised of cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of such traits associated with virulence such as capsule size, urease production and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding for the oligopeptide transporter (CNAG_03013;OPT1) were observed among amoeba-passaged isolates from each of the three strains. In addition, isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene, which encodes Pkr1 (AMP-dependent protein kinase regulator) but were less virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate thatC. neoformanssurvival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes, which confer increase resistance against protozoal predation. Given the myriad of potential predators in soils the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation.Author summaryCryptococcus neoformansis a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. Cryptococcosis is a worldwide concern due to its high mortality rate. A major question in the field is how an environmental yeast such asC. neoformansbecomes a human pathogen when it has no need for animal host in its life cycle. Previous studies showed evidence thatC. neoformansincreases its pathogenicity after interacting with its environmental predator amoebae. Amoebae behave like macrophages, an important immune cell in human body, so it is considered as a training ground for pathogens to resist macrophages. However, howC. neoformanschanges its virulence through interacting with amoebae is unknown. Here, we exposedC. neoformansto amoebae for a long period of time. We found thatC. neoformanscells recovered from amoebae manifested numerous changes to phenotypes related to its virulence and one of the amoeba-passagedC. neoformanscells had enhanced ability to kill macrophages. We further analyzed their genome sequences and found various mutations in different cells of amoeba-passagedC. neoformans, showing that DNA mutations may be the major cause of the phenotypic changes after interacting with amoebae. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival.