Objective: The creatine analogue ß-guanidinopropionic acid (ß-GPA) decreases plasma glucose levels by increasing the sensitivity to insulin. This effect is based on a ß-GPA induced expression of mRNA and total protein content of the insulin-responsive glucose transporter GLUT4. Although the oral availability of ß-GPA is well established, the underlying uptake mechanism has not yet been studied. Methods: We investigated whether the H⁺-coupled amino acid transporter PAT1, which is expressed in the apical membrane of intestinal cells, accepts guanidine derivatives as substrates. Results: Uptake of L-[³H]proline into Caco-2 cells expressing hPAT1 constitutively was strongly inhibited by ß-GPA and its derivatives guanidinoacetic acid (GAA) and 4-guanidinobutyric acid (4-GBA). Competition assays revealed apparent affinity constants in the high affinity range of 1.3, 1.5 and 1.4 mM, respectively, and competitive types of inhibition. Electrophysiological measurements at hPAT1-expressing Xenopus laevis oocytes unequivocally demonstrated that ß-GPA, GAA and 4-GBA are effectively translocated across the cell membrane by this transport system in an electrogenic manner. The compounds elicited currents comparable to those of the hPAT1 prototype substrates glycine and L-proline. For ß-GPA a Michaelis constant of 5.6 ± 0.5 mM was obtained. Conclusion: The orally active antihyperglycemic agent ß-GPA is recognized and transported by the H⁺-coupled amino acid transporter hPAT1. hPAT1 is very likely responsible for the intestinal absorption of the drug allowing its oral administration. Moreover, with ß-GPA, GAA and 4-GBA we identified a new high affinity hPAT1 substrate group that might be an interesting starting point for future investigations.