Intestinal permeability is a critical factor for orally administered drugs. It can be facilitated by uptake transporters or limited by efflux transporters and metabolic enzymes in the intestine. The present study aimed to characterize the Ussing chamber system incorporating human intestinal tissue as an in vitro model for investigating the impact of intestinal uptake/efflux transporters on the intestinal absorption of substrate drugs in humans. We confirmed the functions of major intestinal uptake/efflux drug transporters in freshly isolated human jejunum sections by demonstrating a significant decrease in the mucosal uptake of cefadroxil (peptide transporter 1) and methotrexate (proton-coupled folate transporter), mucosal-to-serosal permeability of ribavirin (concentrative nucleoside transporters/equilibrative nucleoside transporters), and serosal-to-mucosal permeability of P-glycoprotein and breast cancer resistance protein substrates in the presence of their typical inhibitors. The mucosal-to-serosal apparent permeability coefficients (Papp) of 19 drugs, including substrates of drug transporters and cytochrome P450 3A, ranged from 0.60 × 10-6 to 29 × 10-6 cm/s and showed a good correlation with reported fraction of an oral dose that enters the gut wall and passes into the portal circulation with escaping intestinal metabolism (FaFg) values in humans. Furthermore, the Papp values for cefadroxil, methotrexate, and ribavirin in the presence of the corresponding transporter inhibitors underestimated the FaFg of these drugs, which clearly showed that intestinal uptake transporters facilitate their intestinal absorption in humans. In conclusion, the functions of major intestinal uptake/efflux drug transporters could be maintained in freshly isolated human jejunum sections. The Ussing chamber system incorporating human intestinal tissue would be useful for evaluating the impact of intestinal uptake/efflux transporters on the intestinal absorption of various types of drugs in humans. SIGNIFICANCE STATEMENT: Although previous studies have predicted the intestinal absorption of drugs in humans using the Ussing chamber system incorporating human intestinal tissue, there is little systematic information about drug transport mediated by multiple transporters in this system. We confirmed the functions of major intestinal uptake/efflux transporters in freshly isolated human jejunum sections and demonstrated that the mucosal-to-serosal apparent permeability coefficient of various types of drugs showed a good correlation with reported human FaFg values.