We investigated the effects of early exposure to heat stress on total mammary blood flow, trans-mammary disappearance of metabolites and mammary gene expression profiles in lactating dairy cows. Six mid-lactation Holstein cows were housed in tie-stalls in environmental chambers at constant thermoneutrality (TN; THI~65) for 5 d to maintain normal rectal temperature, then exposed to 5 d of programmed constant heat stress (HS; THI~75) to achieve and maintain elevated rectal temperature. Total mammary blood flow was measured throughout the trial by transrectal scanning of both right and left external pudic arteries using color Doppler ultrasound equipped with a convex probe. Mammary arteriovenous blood samples were collected, analyzed for concentration of metabolites, and trans-mammary metabolite disappearance was calculated. Mammary biopsies were obtained from randomly selected, alternating rear mammary glands on d −1 and 5 relative to HS, then mRNA was isolated and sequenced. Genes were considered differentially expressed (DE) when P ≤ 0.002 and FDR ≤ 0.10. Rectal temperature increased whereas feed intake and milk yield decreased (P < 0.001) in HS compared with TN (39.7 vs 38.5 ± 0.1°C, 15.9 vs 22.4 ± 0.4 kg/d, and 29.2 vs 36.7 ± 0.8 kg/d, respectively). HS reduced (P = 0.04) total mammary blood flow (543 vs 466 ± 22 L/h for TN and HS, respectively). Relative trans-mammary disappearance of glucose and triglycerides did not differ between HS and TN, averaging 20.0 ± 1.8% and 44.6 ± 3.5%, respectively. Compared with TN, 402 DE genes were downregulated in HS, which represent functions such as metabolic pathways, carbon metabolism, glycolysis and gluconeogenesis, biosynthesis of amino acids, protein processing in the endoplasmic reticulum, and phagosome, among others. In addition, 452 DE genes were upregulated in HS; annotated functions include focal adhesion, hippo signaling pathway, ECM-receptor interaction, proteoglycans, ribosome, regulation of actin cytoskeleton, and more. Early exposure to heat stress reduced total blood flow and altered gene expression in the mammary gland but did not affect relative nutrient uptake.