Vibrio cholerae, which causes the diarrheal disease cholera, is a species of bacteria commonlyfound in aquatic habitats. Within such environments, the bacterium must defend itselfagainst predatory protozoan grazers. Amoebae are prominent grazers, withAcanthamoebacastellaniibeing one of the best-studied aquatic amoebae. We previously showed thatV.choleraeresists digestion byA. castellaniiand establishes a replication niche within the host’sosmoregulatory organelle. In this study, we decipher the molecular mechanisms involved inthe maintenance ofV. cholerae’s intra-amoebal replication niche and its ultimate escape fromthe succumbed host. We demonstrate that minor virulence features important for disease inmammals, such as extracellular enzymes andflagellum-based motility, have a key role in thereplication and transmission ofV. choleraein its aqueous environment. This work, therefore,describes new mechanisms that provide the pathogen with afitness advantage in its primaryhabitat, which may have contributed to the emergence of these minor virulence factors in thespeciesV. cholerae.