Intestinal alkaline phosphatase (IAP) is the most ancestral of the tissue-specific members of the AP gene family. Several studies have suggested an absorptive function for IAP, but in vivo data to this effect have been lacking. We inactivated the mouse IAP gene in embryo-derived stem cells and generated mice homozygous for the null mutation. The mice were macroscopically and histologically normal and fertile and showed no difference from the wild-type controls under normal laboratory conditions. However, when maintained long-term on a high-fat diet, the IAP-deficient mice showed faster body weight gain than did control animals. Histological examination revealed an accelerated transport of fat droplets through the intestinal epithelium and elevation of serum triglyceride levels in the IAP-deficient mice compared to wild-type mice. Our study suggests that IAP participates in a rate-limiting step regulating fat absorption.