Sulfasalazine is characterized by low intestinal absorption, which essentially enables its colonic targeting and therapeutic action. The mechanisms behind this low absorption have not yet been elucidated. The purpose of this study was to investigate the role of efflux transporters in the intestinal absorption of sulfasalazine, as a potential mechanism for its low small-intestinal absorption, and colonic targeting, following oral administration. The effects of P-gp, MRP2 and BCRP inhibitors on sulfasalazine bidirectional permeability were studied across Caco-2 cell monolayers, including dose-response analysis. Sulfasalazine in-vivo permeability was then investigated in the rat jejunum by single-pass perfusion, in the presence vs. absence of inhibitors. Sulfasalazine exhibited 19-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. MRP2 inhibitors (MK-571 and indomethacin), and BCRP inhibitors (FTC and pantoprazole) significantly increased AP-BL and decreased BL-AP sulfasalazine Caco-2 transport, in a concentration-dependent manner. No effect was observed with the P-gp inhibitors verapamil and quinidine. The IC₅₀ values of the specific MRP2 and BCRP inhibitors MK-571 and FTC on sulfasalazine secretion were 21.5 and 2.0 µM, respectively. Simultaneous inhibition of MRP2 and BCRP completely abolished sulfasalazine Caco-2 efflux. Without inhibitors, sulfasalazine displayed low (vs. metoprolol) in-vivo intestinal permeability in the rat model. MK-571 or FTC significantly increased sulfasalazine permeability, bringing it to the low-high permeability boundary. With both MK-571 and FTC present, sulfasalazine displayed high permeability. In conclusion, efflux transport mediated by MRP2 and BCRP, but not P-gp, shifts sulfasalazine permeability from high to low, thereby enabling its colonic targeting and therapeutic action. To our knowledge, this is the first demonstration of intestinal efflux acting in favor of oral drug delivery.