Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4_114–135 peptide, can overcome these gastrointestinal complications in CF (CFTR^m3Bay null mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR^+/+), heterozygous (CFTR^+/−), or homozygous (CFTR^−/−) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca²⁺ mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I⁻influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR^−/− background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca²⁺or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca²⁺-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4_114–135 to counteract chronic constipation/obstructive bowel syndrome in CF patients.