In order to reach the bloodstream and thus the target receptor, an orally-administered drug must first cross the intestinal barrier, which can occur via a paracellular, passive transcellular, or carrier-mediated uptake and/or efflux process (active or concentration gradient-driven). Our work aimed to explore the transport mechanism of the antiretroviral lamivudine (deoxycytidine nucleoside analogue), using a three-part strategy: in vitro, an ex vivo and an in situ method, represented by PAMPA, rat jejunum patches and rat Single Pass Intestinal Perfusion (SPIP), respectively. The determined permeability coefficients were compared with those from a published Caco-2 and MDCK study. Computational prediction of human jejunal permeability was explored, using various non-human permeability coefficients as descriptors. The ex vivo technique was performed in Franz-type diffusion cells, mounted with male Wistar rat jejunum segment patches. PAMPA was performed with an acceptor solution simulating the binding of serum proteins, an artificial membrane impregnated with egg lecithin/cholesterol and a gradient of pH between donor and acceptor solutions. The SPIP was conducted by proximal jejunum cannulation and drug perfusion in a constant flow rate of 0.2 mL/min. The outcomes of our studies showed the following predicted pattern for lamivudine effective jejunal permeability: P(eff)(exvivoA>B)>P(eff)(SPIP)>P(eff)(exvivo B>A)>P(eff)(Caco-2)≈P(eff)(MDCK)≈P(eff)(PAMPA), strongly suggesting that this compound has carrier-mediated uptake as its dominant transport mechanism. Notwithstanding, Caco-2 cells may indicate an under-expression of uptake transporters and possibly an over-expression efflux transporters, compared to that found in the rat jejunum.