Key points Among the three Shank proteins in human (Shank1–3), Shank2 is abundantly expressed in epithelial cells. However, the in vivo physiological role of Shank2 in epithelial transport remains elusive. The functional activity and expression of cystic fibrosis transmembrane conductance regulator (CFTR) and fluid secretion in the gastrointestinal epithelia were examined in the intestines of Shank2+/+ and Shank2−/− mice using an integrated molecular and physiological approach. Shank2 deletion augmented the CFTR‐mediated short‐circuit current in the mouse colon and profoundly increased the cholera toxin‐induced fluid accumulation in the mouse intestine. Shank2 appears to be a key molecule regulating epithelial fluid secretion. Modulation of Shank2‐mediated function would be helpful to treat diseases associated with hypersecretion or hyposecretion in epithelia. Abstract Shank2 is a PDZ (PSD‐95/discs large/ZO‐1)‐based adaptor that has been suggested to regulate membrane transporting proteins in the brain and epithelial tissues. Here, we report that Shank2 mutant (Shank2−/−) mice exhibit aberrant fluid and ion transport in the intestine. Molecular characterization using epithelial tissues from Shank2+/+ and Shank2−/− mice revealed that a long spliceoform of Shank2 (Shank2E) is predominantly expressed in the pancreatic, renal and intestinal epithelia. In functional assays, deletion of Shank2 increased the cystic fibrosis transmembrane conductance regulator (CFTR)‐dependent short‐circuit currents by 84% (P < 0.05) and 101% (P < 0.05) in the mouse colon and rectum, respectively. Disruption of the CFTR–Shank2–phosphodiesterase 4D protein complex appeared to be mostly responsible for the changes in CFTR activities. Notably, Shank2 deletion profoundly increased cholera toxin‐induced fluid accumulation in the mouse intestine (∼90%, P < 0.01). Analyses with chemical inhibitors confirmed that the hyperactivation of CFTR channel function is responsible for the increased response to cholera toxin. These results suggest that Shank2 is a key molecule that participates in epithelial homeostasis, in particular to prevent overt secretory responses caused by epithelial pathogens.