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.