AbstractSmall peptides can be absorbed by the bovine mammary gland for the synthesis of milk protein, but the absorption mechanism still needs further study. In this study, the role of peptide transporters in small peptide uptake by bovine mammary epithelial cells (BMECs) was studied. First, BMECs were obtained and cultured in a transwell chamber. After 5 days of culture, the FITC‐dextran permeability of the cell layer was detected. Then, 0.5 mM methionyl‐methionine (Met‐Met) was added to the medium of the lower and upper transwell chambers, respectively. The culture medium and BMECs were collected after 24 h of treatment. Liquid chromatography‐mass spectrometry (LC–MS) was used to detect the concentration of Met‐Met in the culture medium. Real‐time PCR was used to detect the mRNA abundance of β‐casein, oligopeptide transporter 2 (PepT2), and small peptide histidine transporter 1 (PhT1) in BMECs. Then, the BMECs were transfected with siRNA‐PepT2 and siRNA‐PhT1, respectively, and the uptake of β‐Ala‐Lys‐N‐7‐amino‐4‐methylcoumarin‐3‐acetic acid (β‐Ala‐Lys‐AMCA) in BMECs was detected. The results showed that, after 5 days of culture, the FITC‐dextran permeability of BMECs was 0.6%, which was significantly lower than that of the control group. The absorption rates of Met‐Met in the culture medium of the upper and lower chambers were 99.99% and 99.95%, respectively. The addition of Met‐Met to the upper chamber significantly increased the mRNA abundance of β‐casein and PepT2. The addition of Met‐Met to the lower chamber significantly improved the mRNA abundance of β‐casein, PepT2, and PhT1. The uptake of β‐Ala‐Lys‐AMCA significantly decreased in BMECs transfected with siRNA‐PepT2. These results suggested that the BMECs were successfully cultured in the transwell chamber and formed a cell layer with little permeability. The small peptides in both the upper and lower chambers of the transwell can be absorbed by BMECs in different ways. PepT2 plays an important role in the uptake of small peptides on both the basal and apical sides of BMECs, and PhT1 may be involved in the uptake of small peptides on the basal side of BMECs. Therefore, the addition of small peptides in dairy cow diets may be an effective dietary manipulation to increase milk protein concentration or yield.