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In drug development, estimating fraction absorbed ( F a ) in man for permeability-limited compounds is important but challenging. To model F a of such compounds from apparent permeabilities ( P app ) across filter-grown Caco-2 cell monolayers, it is central to elucidate the intestinal permeation mechanism(s) of the compound. The present study aims to refine a computational permeability model to investigate the relative contribution of paracellular and transcellular routes to the P app across Caco-2 monolayers of the permeability-limited compound acamprosate having a bioavailability of ∼11%. The P app values of acamprosate and of several paracellular marker molecules were measured. These P app values were used to refine system-specific parameters of the Caco-2 monolayers, that is, paracellular pore radius, pore capacity, and potential drop. The refined parameters were subsequently used as an input in modeling the permeability ( P modeled ) of the tested compounds using mathematical models collected from two published permeability models. The experimental data show that acamprosate P app across Caco-2 monolayers is low and similar in both transport directions. The obtained acamprosate P app , 1.56 ± 0.28 × 10 -7 cm·s -1 , is similar to the P app of molecular markers for paracellular permeability, namely, mannitol (2.72 ± 0.24 × 10 -7 cm·s -1 ), lucifer yellow (1.80 ± 0.35 × 10 -7 cm·s -1 ), and fluorescein (2.10 ± 0.28 × 10 -7 cm·s -1 ), and lower than that of atenolol (7.32 ± 0.60 × 10 -7 cm·s -1 ; mean ± SEM, n = 3-6), while the end-point amount of acamprosate internalized by the cell monolayer, Q monolayer , was lower than that of mannitol. Acamprosate did not influence the barrier function of the monolayers since it altered neither the P app of the three paracellular markers nor the transepithelial electrical resistance (TEER) of the cell monolayer. The P modeled for all the paracellular markers and acamprosate was dominated by the P para component and matched the experimentally obtained P app . Furthermore, acamprosate did not inhibit the uptake of probe substrates for solute carriers PEPT1, TAUT, PAT1, EAAT1, B 0,+ AT/rBAT, OATP2B1, and ASBT expressed in Caco-2 cells. Thus, the P modeled estimated well P para , and the paracellular route appears to be the predominant mechanism for acamprosate P app across Caco-2 monolayers, while the alternative transcellular routes, mediated by passive diffusion or carriers, are suggested to only play insignificant roles.