Zidovudine (AZT) was the first drug approved for the treatment of Acquired Immunodeficiency Syndrome (AIDS) in humans, and although its clinical efficacy has been demonstrated, suboptimal pharmacokinetic aspects still remain a concern. To assess the basis of its highly variable oral bioavailability, this work deals with the study of AZT intestinal absorption by applying the gut sac technique. Permeation through the rat jejunum and ileum segments was analyzed at different drug concentrations and gut regions, with higher apparent permeability coefficients (P(app)) being found for the proximal regions of the small intestine compared to distal ones. Bi-directional permeation assays demonstrated that AZT is subjected to efflux mechanisms in distal regions of small intestine, which are blocked by verapamil (VER), thus demonstrating a P-glycoprotein (P-gp) mediated mechanism. The efficiency of AZT efflux increased in the distal ileum as consequence of exposure to AZT, with the amount of drug permeating from the mucosal to the serosal side diminishing after 35 min. Molecular modeling techniques were applied to analyze the binding mode of AZT to P-gp, which was compared to that of VER and AZT-Ac, a novel prodrug of AZT. The energy required for their solvation was found to constitute a critical feature in their binding to this efflux protein. The present work updates the impact of P-gp in AZT oral bioavailability, highlighting the need for further study of the dynamic nature of its expression at intestinal level.