Peptide transporters, also named proton-coupled oligopeptide transporters (POTs), play a great role in mediating cellular uptake of di/tripeptides and peptidomimetic drugs via the inwardly directed proton motive force. However, the expression profile and transport kinetics of the transporters in bovine mammary gland remain unknown. Polymerase chain reaction (PCR) and Western blotting were used to investigate expression of POTs (PepT1, PepT2, PhT1, PhT2) in BMECs. Immunofluorescence was used to determine the location of the peptide transporters in bovine mammary gland tissue. Uptake and transport kinetic studies were performed with stable metabolically β-alanyl-l-lysyl-Nε-7-amino-4-methyl-coumarin-3-acetic acid (β-Ala-Lys-AMCA) in BMECs. The effects of time, pH, concentration and inhibitors on β-Ala-Lys-AMCA uptake were studied. All experiments were performed in 3 replicates. Data were analyzed using GLM procedure of SAS software. The results showed PepT2 and PhT1 are expressed in bovine mammary gland by using PCR and Western blotting. The immunofluorescence results showed that PhT1 and PepT2 are both located on the apical membrane and basolateral membrane in BMECs. The uptake of β-Ala-Lys-AMCA was linear during the 60 min incubation period and reached plateau at 60 min. The optimal pH for the uptake of β-Ala-Lys-AMCA in BMECs was 6.5. The transport kinetic study suggested that the uptake of β-Ala-Lys-AMCA is saturable over the tested concentration. The binding affinity (Km) and the maximal velocity (Vmax) exhibited in BMECs is 82 µmol and 124 pmol/min/mg protein. The competitive inhibition result showed Gly-Sar, Met-Gly and Met-Met significantly inhibited β-Ala-Lys-AMCA uptake. However, histidine had no effect on β-Ala-Lys-AMCA uptake indicating that PhT1 may not be involved in β-Ala-Lys-AMCA uptake. Furthermore, knockdown of PepT2 significantly inhibited β-Ala-Lys-AMCA uptake. The above results showed that PepT2 may be involved in peptide transport, but PhT1 may not function in peptide transport in bovine mammary gland.