Background <p>This study was conducted to investigate the impact of varying degrees of heat stress on milk protein synthesis in dairy cows using comprehensive analyses of metabolomics and proteomics. Eighteen dairy cows were subjected to no heat stress (No-HS), mild heat stress (Mild-HS), and moderate heat stress (Mod-HS). Blood and milk samples were collected to determine the content and composition of amino acids (AA), and milk samples were used for metabolomic and proteomic analyses.</p> Results <p>Milk protein yield was significantly lower under Mild-HS and Mod-HS than No-HS (<i>P</i> &lt; 0.001). During Mild-HS, no significant difference was found in total AA concentration in both arterial (<i>P</i> = 0.545) and venous blood (<i>P</i> = 0.057), but arterial AA supply to the mammary gland significantly increased (<i>P</i> = 0.045) when compared with No-HS. Under Mod-HS, the supply (<i>P</i> &lt; 0.001) and uptake (<i>P</i> = 0.001) of total AA in the mammary gland decreased significantly, affecting the availability of precursors necessary for milk protein synthesis. Milk metabolomic analysis revealed that Mod-HS significantly impacted nucleotide metabolism, energy metabolism, and protein synthesis processes including translation, folding, and transport. Milk proteomic analysis showed significant downregulation of ribosomal and heat shock proteins which are crucial for protein synthesis and folding.</p> Conclusions <p>These findings suggest that heat stress disrupts AA utilization and energy metabolism in the mammary gland, leading to the reduced efficiency in milk protein synthesis and lowered milk protein yield. This study offers valuable insights into the metabolic and proteomic changes in dairy cows under heat stress, highlighting potential strategies to mitigate the adverse effects of heat stress on dairy production and milk quality.</p>