To elucidate mechanisms involved in M cell uptake and transcytosis of Vibrio cholerae, we used an in vitro model of human M-like cells in a Caco-2 monolayer. Interspersed among the epithelial monolayer of Caco-2 cells we detect cells that display M-like features with or without prior lymphocyte treatment and we have established key parameters for V. cholerae transcytosis in this model. Cholera toxin (CT) mutants lacking the A subunit alone or both the A and B subunits were deficient for transcytosis. We explored this finding further and showed that expression of both subunits is required for binding by whole V. cholerae to immobilized CT receptor, the glycosphingolipid GM1. Confocal microscopy showed CT associated with transcytosing bacteria, and transcytosis was inhibited by pre-incubation with GM1 before infection. Finally, heat treatment of the bacterial cells caused a loss of binding to GM1 that was correlated with a significant decrease in uptake and transcytosis by the monolayer. Our data support a model in which the ability of bacteria to interact with GM1 in a CT-dependent fashion plays a critical role in transcytosis of V. cholerae by M cells.