The absorption of drugs from the gastrointestinal tract is one of the important determinants for oral bioavailability. Development of in vitro experimental techniques such as isolated membrane vesicles and cell culture systems has allowed us to elucidate the transport mechanisms of various drugs across the plasma membrane. Recent introduction of molecular biological techniques resulted in the successful identification of drug transporters responsible for the intestinal absorption of a wide variety of drugs. Each transporter exhibits its own substrate specificity, though it usually shows broad substrate specificity. In this review, we first summarize the recent advances in the characterization of drug transporters in the small intestine, classified into peptide transporters, organic cation transporters and organic anion transporters. In particular, peptide transporter (PEPT1) is the best-characterized drug transporter in the small intestine, and therefore its utilization to improve the oral absorption of poorly absorbed drugs is briefly described. In addition, regulation of the activity and expression levels of drug transporters seems to be an important aspect, because alterations in the functional characteristics and/or expression levels of drug transporters in the small intestine could be responsible for the intra- and interindividual variability of oral bioavailability of drugs. As an example, regulation of the activity and expression of PEPT1 is summarized.