The current study was planned to evaluate the effects of heat stress (HS) on mammary blood flow and the profile of circulating amino acids (AA) in blood and milk of lactating Holstein dairy cows. For this purpose, twenty dairy cows with similar body conditions, parity, and milk production were divided into two treatments of 10 animals each. The first treatment group was exposed to HS and reared in summer season, and second treatment group were reared in thermoneutral environment (TN) during spring season. Results showed that HS resulted in predominantly high level of total AA (TAA), glycine, serine (Ser), alanine (P<0.05) while predominant decrease concentrations of methionine (Met) and tryptophan (Trp) (P<0.05) in external pudic artery and mammary vein of experimental cows. Methionine, arginine (Arg), Trp and threonine (Thr) (P<0.05) supply to the mammary gland by pudic artery and lower amount of Met and Trp in mammary vain output (P<0.05) was observed in HS cows. Similarly, the uptake of most AA in the udder was decreased due to HS (P<0.05) and resulted in a lower (P<0.05) amount of TAA, glutamine, phenylalanine, Met, lysine, leucine, tyrosine, isoleucine, Ser, Thr, valine, and histidine in milk. A highly significant decrease (P<0.01) in amount of Arg and Trp in milk was observed in HS cows. It was concluded that during HS blood AA use in dairy animals is shifted away from milk protein synthesis by lower mammary blood flow and AA uptake by mammary gland.