The objective of this study was to establish a primary culture of omasal epithelial cells (OECs) derived from dairy calves and to characterize its function in small peptide absorption. Bovine omasal tissues were obtained from newborn Chinese Holstein calves and digested with a 2.5% trypsin solution to obtain OECs. The isolated cells were later cultured in DMEM containing 10% fetal bovine serum, 5 μg/ml insulin, 10 ng/ml epidermal growth factor, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamycin and 2.5 μg/ml amphotericin B. Hematoxylin and eosin staining of omasal tissue after digestion indicated that the cultured cells originated from the epithelial strata. Pure epithelial cells displayed an epithelial cell-like morphology, similar to cobblestone, with few visible fibroblasts and were cytokeratin 18-positive according to immunocytochemical analyses. The OECs were morphologically characterized with desmosomes, tight junctions and microvilli. These cells exhibited normal growth properties, as assessed using a cell growth curve, and were stably cultured for 10 passages. The OECs expressed the peptide transporter 1 (PEPT1) mRNA and absorbed intact glycylsarcosine (Gly-Sar). The uptake of Gly-Sar by OECs was pH-dependent with an optimal pH of 5.5-6.5. Furthermore, the uptake of Gly-Sar was also time-dependent, concentration-dependent and temperature-dependent. Moreover, PEPT1 was saturated with Gly-Sar at a concentration of 2.5 mM. The uptake via PEPT1 was higher compared with that via passive route at low substrate concentrations (<1.5 mM). This result suggested that PEPT1 contributed more to total small peptide absorption at low concentrations. In addition, this uptake could be competitively inhibited by methionine-glycine. Taken together, these data suggested that PEPT1 contributes to small peptide absorption in OECs. Thus, OECs may serve as a useful culture model for the study of the absorption of small peptides in bovine omasum.