The synthesis of milk protein requires the availability of amino acids and a large supply of energy. Lysine, methionine and glucose regulate milk protein production of lactating dairy cows in vivo. Moreover, the content and ratio of lysine to methionine can regulate milk protein synthesis via cellular signaling pathways involving JAK2-STAT5 and mammalian target of rapamycin (mTOR). This study was conducted to investigate the effects of different lysine/methionine pattern and glucose level on milk protein synthesis in vitro, mainly focused on genes related to JAK2-STAT5 and mTOR. Primary bovine mammary epithelial cells (BMEC) were obtained from Holstein dairy cows and cultured in Dulbecco's modified Eagle's medium-F12 medium containing 10% fetal bovine serum. BMEC were subjected to 4 treatments arranged in a 2 × 2 factorial design with lysine/methionine ratio (3: 1 vs. 2.3: 1, namely balanced vs. unbalanced) and glucose level (17.5 mM vs. 2.5mM, namely high vs. low) as 2 factors. In this experiment, total casein content, cell proliferation and genes expression related to JAK2-STAT5 and mTOR pathways were measured. Compared with low level of glucose groups, casein content and cell proliferation increased in groups with high level of glucose (P < 0.05). Casein content was also higher in Lysine/Methionine balanced groups than in unbalanced groups (P < 0.01). Expression of CSN1S2, CSN2, LALBA, STAT5, ELF5, mTOR (P < 0.01) and CSN1S1 (P < 0.05) were upregulated in groups with high level of glucose compared with low level groups. The upregulation of CSN1S2, CSN2, LALBA, JAK2, STAT5, ELF5, mTOR (P < 0.01) and CSN1S1, CSN3 (P < 0.05) were also observed in Lysine/Methionine balanced groups compared with unbalanced groups, while EIF4EBP1 was downregulated (P < 0.05). In conclusion, proper ratio of Lysine to methionine and high level of glucose may directly accelerate the BMEC proliferation and regulate the expression of genes related to milk protein transcription and translation, which can increase milk protein synthesis.