Because O blood group has been associated with more severe cholera infections, it has been hypothesized that cholera toxin (CT) may bind non-O blood group antigens of the intestinal mucosae, thereby preventing efficient interaction with target GM1 gangliosides required for uptake of the toxin and activation of cyclic adenosine monophosphate (cAMP) signaling in target epithelia. Herein, we show that after exposure to CT, human enteroids expressing O blood group exhibited marked increase in cAMP relative to cells derived from blood group A individuals. Likewise, using CRISPR/Cas9 engineering, a functional group O line (HT-29-A−/−) was generated from a parent group A HT-29 line. CT stimulated robust cAMP responses in HT-29-A−/− cells relative to HT-29 cells. These findings provide a direct molecular link between blood group O expression and differential cellular responses to CT, recapitulating clinical and epidemiologic observations.