Abstract: The analgesic effect of tramadol (TMD) results from the monoaminergic effect of its two enantiomers, (+)-TMD and (−)-TMD as well as its opioid metabolite (+)-O-desmethyl-tramadol (M1). P-glycoprotein (P-gp) might be of importance in the analgesic and tolerability profile variability of TMD. Our study investigated the involvement of P-gp in the transepithelial transport of (+)-TMD, (−)-TMD and M1, using a Caco-2 cell monolayer model. The bidirectional transport of racemic TMD and M1 (1–100 µM) across the monolayers was investigated at two pH conditions (pH 6.8/7.4 and 7.4/7.4) in the presence and absence of P-gp inhibitor cyclosporine A (10 µM) and assessed with the more potent and specific P-gp inhibitor GF120918 (4 µM). Analytical quantification was performed by liquid chromatography coupled to the fluorescence detector. A net secretion of (+)-TMD, (−)-TMD and M1 was observed when a pH gradient was applied (TR: Papp(B − A)/Papp(A − B): 1.8–2.7; P < 0.05). However, the bidirectional transport of all compounds was equal in the non-gradient system. In the presence of P-gp inhibitors, a slight but significant increase of secretory flux was observed (up to 26%; P < 0.05) at both pH conditions. In conclusion, (+)-TMD, (−)-TMD and M1 are not P-gp substrates. However, proton-based efflux pumps may be involved in limiting the gastrointestinal absorption of TMD enantiomers as well as enhancing TMD enantiomers and M1 renal excretion. A possible involvement of uptake carriers in the transepithelial transport of TMD enantiomers and M1 is suggested.