Knockout mouse models have provided key insights into the physiological significance of many intestinal electrolyte transporters. This review has selected three examples to highlight the importance of knockout mouse technology in unravelling complex regulatory relationships important for the understanding of human diseases. Genetic ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) has created one of the most useful mouse models for understanding intestinal transport. Recent work has provided an understanding of the key role of the CFTR anion channel in the regulation of HCO₃⁻ secretion, and the important consequences that a defect in HCO₃⁻ output may have on the viscoelastic properties of mucus, on lipid absorption and on male and female reproductive function. The regulation of CFTR activity, and also that of the intestinal salt absorptive transporter NHE3, occurs via the formation of PSD95‐Drosophila homologue Discs‐large‐tight junction protein ZO‐1 (PDZ) adaptor protein‐mediated multiprotein complexes. The recent generation of knockout mice for three members of the sodium‐hydrogen regulatory factor (NHERF) family of PDZ adaptor proteins, namely NHERF1 (EBP50), NHERF2 (E3KARP) and NHERF3 (PDZK1), has helped to explain why NHERF1 is essential for both normal and mutant CFTR function. In addition, they have provided new insight into the molecular mechanisms of secretory diarrhoeas. Genetic ablation of members of the recently discovered Slc26 anion transporter gene family not only reproduced the phenotype of the genetic diseases that led to the discovery of the gene family, but also resulted in new insights into complex human diseases such as secretory diarrhoea, fructose‐induced hypertension and urolithiasis.