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The present study aimed at investigating whether increasing concentrations of glucose supply have a depressive effect on the mRNA abundance and activity of key gluconeogenic enzymes in dairy cows. Twelve Holstein-Friesian dairy cows in mid-lactation were intravenously infused with saline (SI; n = 6) or a 40% glucose solution (GI; n = 6). For GI cows, the infusion dose increased by 1.25%/d relative to the initial NE(l) requirement until a maximum dose equating to surplus 30% NE(l) was reached on d 24. Cows receiving SI received an equivalent volume of 0.9% saline solution. Blood samples were taken every 2 d, and liver biopsies were collected every 8 d. A treatment x quadratic dose interaction (P < 0.01) was observed for the concentration of plasma glucose and serum insulin. The interactions were due to positive quadratic responses of the concentrations of glucose and insulin for GI cows, whereas the concentrations of glucose and insulin did not change over time for SI cows. The concentration of beta-hydroxybutyric acid (BHBA) and serum urea nitrogen (BUN) responded in a treatment x quadratic dose manner, such that greater decreases (P < 0.01) in BHBA and BUN concentrations were observed for cows receiving GI than SI as the dosage increased. Serum NEFA concentration tended to follow a similar pattern as serum BHBA and BUN; however, the interaction was not significant (P = 0.07). The mRNA abundance of gluconeogenesis enzymes followed a linear treatment x dose interaction (P < 0.05) for only pyruvate carboxylase (PC), which was paralleled by a trend for a linear treatment x dose interaction (P = 0.13) for PC enzyme activity. The least PC expression and activity were observed at the largest glucose dosage. The activity, but not mRNA abundance, of fructose 1,6-bisphosphatase (FBPase) showed treatment x quadratic dose interactions (P < 0.05) with decreasing activity at increasing glucose dose. Activities and expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase were not affected (P > 0.25) by treatment. In conclusion, hepatic gluconeogenic enzymes are only moderately affected by slowly increasing glucose supply, including a translational or posttranslational downregulation of FBPase activity and a decrease in the mRNA abundance of PC with possible consequences for PC enzyme activity.