While chronic stress can decrease milk production, an acute, high-dose of a synthetic glucocorticoid such as dexamethasone (DEX) can abruptly and transiently suppress milk yield. The mechanism underlying this response, and whether it is realized systemically or locally within the mammary gland, has not been established. We sought to define the effect of acute DEX on the yield and composition of milk produced by dairy cows and the accompanying changes in mammary gland gene expression. A single, high dose of DEX (40 mg, IM) was administered to 4 multiparous lactating Holstein cows (55.7 ± 5.3 DIM, 746 kg average body weight). Mammary gland biopsies were performed on alternating quarters at 0, 12, 24, and 72 h post-DEX. To define changes in milk volume and composition, cows were quarter-milked every 12 h for 7 d before through 5 d after DEX. Oxytocin (IV, 5 IU) was administered after each milking to ensure complete milkout, and milk samples were collected both before and after oxytocin. Milk was analyzed for lactose, protein, fat, casein, and solids. Total RNA was isolated from biopsied mammary tissue and subjected to 3′-TAG RNA sequencing for differential gene expression analysis. Data were analyzed with a mixed effects repeated measures ANOVA. Within 12 h of DEX, average 12-hourly milk yield decreased from 29.1 to 22.5 kg (P < 0.05) and returned to baseline by 72 h. Lactose concentration decreased from 4.96% at 0 h to 4.84% at 72 h (P < 0.05). There were no differences in the concentration of casein, fat, total protein, or solids in milk. Based on 3′-TAG RNA sequencing data, α-lactalbumin (LALBA) gene expression was suppressed within 12 h of DEX (P < 0.05). Complete differential gene expression and KEGG pathway analyses will define which genes and pathways are stimulated or suppressed in the lactating mammary gland in response to DEX. These data show that DEX induced a transient suppression of milk production, LALBA expression, and lactose production, without affecting fat, total protein, or casein content.