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The underlying molecular mechanisms that control milk yield and milk protein yield in domestic animals are not completely understood. In this study, the galactopoietic response to exogenous growth hormone (GH) was used as an experimental model to investigate the role of translation initiation and elongation in the regulation of milk protein synthesis in the mammary gland. A slow-release formula of commercially available GH was administered via a single subcutaneous injection to 4 lactating cows (GH group). A further 4 cows were given a single subcutaneous injection of saline (control group). Changes in mRNA transcript level and protein phosphorylation status of key members of the mammalian target of rapamycin (mTOR) pathway were assessed in mammary gland tissues of these animals using quantitative real-time PCR and Western blotting. The GH treatment enhanced the phosphorylation of ribosomal protein S6 and increased the protein abundance of eukaryotic initiation factor 4E (eIF4E) and eukaryotic elongation factor 2 (eEF2) proteins in the mammary gland of GH-treated animals. These results indicate a link between milk protein synthesis and the regulation of mRNA translation. The GH treatment did not change mRNA abundance of ribosomal protein S6, eIF4E, and eEF2, nor did it change the mRNA (mTOR, eEF2 kinase) or protein abundance of eEF2 kinase. These results demonstrate that GH administration changes mRNA translation initiation and elongation possibly via the mTOR pathway (suggested by the increased levels of ribosomal protein S6 phosphorylation), indicating that the mTOR pathway might be a potential control point in the regulation of milk protein synthesis in the mammary gland.