Although oral drug administration is currently the favorable route of administration, intestinal drug absorption is challenged by several highly variable and poorly predictable processes such as gastrointestinal motility, intestinal drug solubility and intestinal metabolism. One further determinant identified and characterized during the last two decades is the intestinal drug transport that is mediated by several transmembrane proteins such as P-gp, BCRP, PEPT1 and OATP2B1. It is well-established that intestinal transporters can affect oral absorption of many drugs in a significant manner either by facilitating their cellular uptake or by pumping them back to gut lumen, which limits their oral bioavailability. Their functional relevance becomes even more apparent in cases of unwanted drug-drug interactions when concomitantly given drugs that cause transporter induction or inhibition, which in turn leads to increased or decreased drug exposure. The longitudinal expression of several intestinal transporters is not homogeneous along the human intestine, which may have functional implications on the preferable site of intestinal drug absorption. Besides the knowledge about the expression of pharmacologically relevant transporters in human intestinal tissue, their exact localization on the apical or basolateral membrane of enterocytes is also of interest but in several cases debatable. Finally, there is obviously a coordinative interplay of intestinal transporters (apical–basolateral), intestinal enzymes and transporters as well as intestinal and hepatic transporters. This review aims to give an updated overview about the expression, localization, regulation and function of clinically relevant transporter proteins in the human intestine.