The molecular and cellular mechanisms underlying the adaption of the mammary gland (MG) to increased ambient heat, and which account for changes in milk composition and immunity are not known yet. This study aimed to elucidate the expression of MG proteins and differences in milk composition and ruminal short-chain fatty acid concentrations of dairy cows experiencing 7 days of heat stress (28°C, THI = 76), pair-feeding (PF) or ad libitum feeding (CON) at thermoneutrality (16°C, THI = 60; each n=10). The feed intake of PF cows were matched to the intake of HS cows. The portion of ruminal acetate, the acetate:propionate ratio, and milk urea concentration were greater whereas milk protein yield and lactose yield were lower in HS than CON cows on day 3 and 6 of the challenge, whilst energy and fat corrected milk were lower in HS than CON and PF cows. The proteome analysis revealed induced bacterial invasion of epithelial cells, leukocyte transendothelial migration, reduction of the pyruvate and carbon metabolism, and platelet activation in the MG of HS compared to CON and/or PF cows. These results highlight adaptive metabolic and immune responses to mitigate the negative effects of ambient heat in the mammary gland.