Leucine, as a signal regulator, could regulate milk casein synthesis by mammalian target of rapamycin (mTOR) signaling pathway. It is transported into cell via L system amino acid transporter 1 (LAT1) accompanying the output of glutamine. However, the upstream signals in the mTOR activation by leucine are still unclear in bovine mammary epithelial cells (BMECs). This study investigated whether LAT1 and leucyl-tRNA synthetase (LARS)-induced mTOR signaling could regulate κ-casein synthesis in BMECs. The BMECs were established by enzymatic digestion of mammary tissue from mid-lactation cows. According to the cell viability, the mRNA expression of LAT1 and LARS in medium lacking leucine and glutamine, 12 h post-starvation was considered the optimal starvation time to determine the cellular signal of leucine influence on κ-casein synthesis with a deficiency of leucine or glutamine. Passage 2 mammary epithelial cells were divided into 4 groups, including the entire AA profile (CTL), leucine deficiency (CTL−Leu, −Leu), glutamine deficiency (CTL−Gln, −Gln), and deficiency of both leucine and glutamine (CTL−Leu−Gln, −Leu−Gln) treatments. The LAT1, ASC system amino acid transporter 2 (ASCT2), LARS, mTOR, ribosomal protein S6 kinase 1 (S6K1) proteins, and the κ-casein were determined. All data were analyzed using one-way ANOVA by SPSS 16.0 (SPSS Inc., Chicago, IL). The results showed that compared with the CTL treatment, LAT1 and LARS proteins were significantly decreased in −Leu and –Leu−Gln treatments (P < 0.05). The phosphorylation mTOR was significantly decreased by the −Gln and –Leu−Gln treatments (P < 0.05), and there was a similar trend (P = 0.057) in the −Leu treatment. The phosphorylation of S6K1 was significantly decreased in −Leu, −Gln and –Leu−Gln compared with CTL and later reduced the κ-casein protein expression (P < 0.05). These results suggested that κ-casein synthesis was affected by leucine stimulated mTOR signaling pathway via activating LAT1 and LARS in BMECs.