Chlorogenic acid (CGA), as a polyphenol abundant in daily diet, exerted various intestinal functions. However, the material basis of its intestinal bioactivities was still unclear. Our study investigated the gastrointestinal distribution of CGA using ultra high performance liquid chromatography-tandem mass spectrum (UPLC-MS/MS) and explored the main drivers leading to different metabolic fates of the metabolites. After oral administration of 50 mg/kg bw CGA to mice, CGA and sulfated metabolites were mainly determined in small intestine. Protocatechuic acid, catechol, and 3-hydroxyphenylpropionic acid were found in copious amounts in large intestine and feces. Besides, caffeic acid existed in the entire intestine and feces. In the in vitro mice/human fecal fermentation of CGA, the production of metabolites was consistent with that in the colon, indicating that microorganisms might lead to the difference in the gastrointestinal distribution of CGA metabolites. Moreover, CGA, 3-hydroxyphenylpropionic acid, protocatechuic acid, catechol, and caffeic acid reduced proinflammatory cytokines (interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)) and increased mucin (Muc2) and tight junction (occludin) mRNA in LPS-induced Caco-2/HT-29 MTX co-culture cells. In conclusion, the biotransformation of CGA in different gastrointestinal tracts varied significantly, and its metabolites could partially support its intestinal bioactivities.