The peptide bound amino acids were shown to promote milk protein synthesis more efficiently than free amino acids. However, the underlying mechanism remains unknown. The aim of this study was to investigate the effect of methionyl-methionine (Met-Met) on milk protein synthesis and to elucidate the underlying mechanism of Met-Met regulating milk protein synthesis in primary bovine mammary gland epithelial cells (BMECs). The BMECs were treated with different concentrations of Met-Met (0, 20, 40, 80, 120, 160 µg/mL). In some experiments, the cells were treated with mTOR inhibitor (rapamycin, 100 ng/mL). The protein expression of mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase 1 gene (S6K1), eukaryotic initiation factor 4E binding protein 1 gene (4E-BP1), β-casein (β-CN), peptide transporter 2 (PepT2), peptide histidine transporter (PhT1) and cyclin D1 were further verified by Western blot. Data were analyzed by GLM procedure of SAS software (SAS Institute, USA). Compared with the control group (addition of 0 µg/mL Met-Met), the expression of β-CN were significantly increased by supplementation of 80 µg/mL Met-Met, but were significantly decreased when mTOR was inhibited by rapamycin. Cyclin D1 and the cell viability were also enhanced by supplementation of 80 µg/mL Met-Met and the inhibition of mTOR suppressed Met-Met-promoted cell proliferation. The expression of PepT2 was significantly enhanced by addition of 80 µg/mL Met-Met, However, the expression of PhT1 was not affected by 80 µg/mL Met-Met. The inhibition of PepT2 by siRNA decreased Met-Met stimulated β-CN expression. The addition of 80 µg/mL Met-Met promoted phosphorylation of mTOR, S6K1 and 4E-BP1. The results suggested Met-Met is absorbed by PepT2 and then Met-Met improve β-CN synthesis by enhancing cell proliferation and activating mTOR signaling pathway through mTOR signaling pathway in BMECs.