The interaction of the antibacterial phosphonodipeptide alafosfalin with mammalian H+/peptide cotransporters was studied in Caco-2 cells, expressing the low-affinity intestinal type peptide transporter 1 (PEPT1), and SKPT cells, expressing the high-affinity renal type peptide transporter 2 (PEPT2). Alafosfalin strongly inhibited the uptake of [14C]glycylsarcosine with Ki values of 0.19 ± 0.01 mm and 0.07 ± 0.01 mm for PEPT1 and PEPT2, respectively. Saturation kinetic studies revealed that in both cell types alafosfalin affected only the affinity constant (Kt) but not the maximal velocity (Vmax) of glycylsarcosine (Gly-Sar) uptake. The inhibition constants and the competitive nature of inhibition were confirmed in Dixon-type experiments. Caco-2 cells and SKPT cells were also cultured on permeable filters: apical uptake and transepithelial apical to basolateral flux of [14C]Gly-Sar across Caco-2 cell monolayers were reduced by alafosfalin (3 mm) by 73%. In SKPT cells, uptake of [14C]Gly-Sar but not flux was inhibited by 61%. We found no evidence for an inhibition of the basolateral to apical uptake or flux of [14C]Gly-Sar by alafosfalin. Alafosfalin (3 mm) did not affect the apical to basolateral [14C]mannitol flux. Determined in an Ussing-type experiment with Caco-2 cells cultured in Snapwells™, alafosfalin increased the short-circuit current through Caco-2 cell monolayers. We conclude that alafosfalin interacts with both H+/peptide symporters and that alafosfalin is actively transported across the intestinal epithelium in a H+-symport, explaining its oral availability. The results also demonstrate that dipeptides where the C-terminal carboxyl group is substituted by a phosphonic function represent high-affinity substrates for mammalian H+/peptide cotransporters.