Vibrio cholerae is the causative agent of the deadly diarrheal disease cholera. As part of its life cycle, V. cholerae persists in marine environments, where it forms surface-attached communities commonly described as biofilms. Evidence indicates that these biofilms constitute the infectious form of the pathogen during outbreaks. Previous work has shown that biofilm-derived V. cholerae cells, even when fully dispersed from the biofilm matrix, are vastly more infectious than planktonic (free-living) cells. Here, we sought to identify factors that contribute to biofilm-induced hyperinfectivity in V. cholerae, and we present evidence for one aspect of the molecular basis of this phenotype. We identified proteins upregulated during growth in biofilms and determined their contributions to the hyperinfectivity phenotype. We found that PstS2, the periplasmic component of the Pst2 phosphate uptake system, was enriched in biofilms. Another gene in the pst2 locus was transcriptionally upregulated in biofilms. Using the infant mouse model, we found that mutation of two pst2 components resulted in impaired colonization. Importantly, deletion of the Pst2 inner membrane complex caused a greater colonization defect after growth in a biofilm compared to shaking culture. Based on these data, we propose that V. cholerae cells in biofilms upregulate the Pst2 system and therefore gain an advantage upon entry into the host. Further characterization of factors contributing to biofilm-induced hyperinfectivity in V. cholerae will improve our understanding of the transmission of the bacteria from natural aquatic habitats to the human host.