Food would seem to be one of the main ways of animal and human contamination with polycyclic aromatic hydrocarbons (PAHs). In vivo studies suggest a transfer in intestinal epithelium by diffusion, which appears extensively governed by the physicochemical properties of PAHs, particularly lipophilicity. However, other mechanisms, such as metabolism, are considered to intervene. Our work aimed at testing in vitro intestinal metabolism and defining its impact on transepithelial transport of PAHs. Caco-2 cells were cultivated on permeable filters and incubated with 14C-labeled benzo[a]pyrene (BaP), pyrene (Pyr), and phenanthrene (Phe), which differ in their physicochemical properties. The results showed that the cells were able to metabolize the compounds. In basal media, Phe appeared to be the least hydroxylated molecule (45% after a 6-h exposure), followed by Pyr (65%) and finally BaP (96%). Inhibition of PAH metabolism showed a determinant effect on kinetics profiles. Transfer in the basal compartment of BaP, Pyr, and Phe radioactivities was, respectively, 26, 4, and 2 times lower with inhibitors, corroborating that intestinal metabolism of PAHs would have a positive impact on their transfer, an impact that increased with their lipophilicity. Furthermore, after a 6-h incubation, metabolites were also detected in apical medium. These findings suggested that intestinal metabolism might play a key role in intestinal barrier permeability and thus in the bioavailability of tested micropollutants.