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 (P) of 19 drugs, including substrates of drug transporters and cytochrome P450 3A, ranged from 0.60 × 10 to 29 × 10 cm/s and showed a good corre