Assessing the regulatory effect of individual amino acids (AA) on milk protein synthesis is vital to AA requirement models for lactation. This study employed the immortalized bovine mammary epithelial cells (CMEC-H) as a model to investigate the effects of Leu or His on mTOR signaling and casein synthesis by Western blotting. Cells were cultured in the Earle's balanced salts (EBSS) with Leu (0.45, 1.35, 5.4, 10.8 mmol/L) or His (0.15, 1.2, 4.8, 9.6 mmol/L) addition for 6h, respectively, and the EBSS without AA was set as negative control, the EBSS supplemented with 10% FBS was used as positive control. The protein band values from the AA-supplemented cells were related to their AA-deprived controls. The experimental data were analyzed using the Duncan’s test for post-hoc multiple comparisons of treatment means by SAS. Meaningful relationships among phosphorylation of the signaling proteins and casein expression were quantified with simple linear regression models using the REG procedure of SAS. Differences between experimental groups were considered significant at a P < 0.05. The results showed that, compared with the negative control, Leu or His significantly increased the phosphorylation of mammalian target of rapamycin (mTOR, Ser2481), a binding partner of target of rapamycin (raptor, Ser792), the ribosomal protein S6 kinase 1 (S6k1, Thr389), eukaryotic initiation factor 4E (eIF4E, Ser209), eukaryotic elongation factor 2 (eEF2, Thr56) and casein synthesis (P < 0.05). These results suggest that the supplement of Leu or His could activate the mTOR pathway and in turn catalyze the phosphorylation of signaling protein and increase milk protein synthesis. Our linear regression model assay declared that the expression of αs1-casein was positively correlated with P-mTOR (R² = 0.7820, P < 0.01), P-S6k1, (R² = 0.7881, P < 0.01) and eEF2 (R² = 0.7835, P < 0.01) with a dose-dependent effect of Leu. While the expression of β-casein (R² = 0.9638, P < 0.01) and κ-casein (R² = 0.9048, P < 0.01) were positively correlated with P-eEF2 with a dose-dependent effect of His. In conclusion, our results can provide certain basic information for the further study of the regulation mechanism of Leu or His on casein expression via mTOR pathway in CMEC-H.