Valine, a kind of branched-chain amino acid, plays a regulatory role beyond that of a building block in milk protein synthesis. However, the underlying molecular mechanism through which valine stimulates β-casein synthesis has not been clarified. Therefore, our study aimed to evaluate the effect of valine on β-casein synthesis and shed light into the molecular mechanism using an model. Results showed that valine supplementation significantly increased β-casein synthesis in bovine mammary epithelial cells (BMECs). Meanwhile, the supplementation of valine resulted in high levels of branched-chain aminotransferase transaminase 2 (BCAT2), TCA-cycle intermediate metabolites, and ATP, AMP-activated protein kinase (AMPK) inhibition, and mammalian target of rapamycin (mTOR) activation. Furthermore, the inhibition of BCAT2 decreased the β-casein synthesis and downregulated the AMPK-mTOR pathway, with similar results observed for AMPK activation. Together, the present data indicate that valine promotes the synthesis of β-casein by affecting the AMPK-mTOR signaling axis and that BCAT2-mediated valine catabolism is the key target.