Updated project metadata. The gastrointestinal tract is covered by a single layer of epithelial cells that, together with the mucus layers, protect the underlying tissue from bacterial invasion. The epithelium has one of the highest turnover rates in the body, renewing every 4-5 days. Using stable isotope labelling, high-resolution mass spectrometry and computational analysis, we report here a comprehensive dataset of the turnover rate of 3041 and the expression of 5012 intestinal epithelial cell proteins, analyzed under conventional and germ-free conditions across five different segments in mouse intestine. The median protein half-life was shorter in small intestine compared to colon, ranging from 3.5 to 4.2 days. Differences in protein turnover rates along the intestinal tract can be explained by distinct physiological functions and site-specific immune responses between the small and large intestine. Absence of microflora resulted in increased protein half-life by approximately one day.