Hyperketonemia (HYK) has negative effects on dairy cattle production and health and may shift nutrient utilization. The objective was to determine if there was a difference in fatty acid (FA) uptake by the mammary gland of Holstein cows with and without HYK (nonHYK) and if those differences are reflected in FTIR predicted milk FA. Blood was sampled at the tail (representing arterial blood) and mammary vein after the a.m. milking twice within 25 d postpartum. A corresponding milk sample was analyzed by FTIR for components and predicted FA. Cows were diagnosed as HYK (≥1.2 mM BHB) or nonHYK on the day of sampling resulting in 9 HYK (1.4 mM+SE BHB) and 12 nonHYK (0.7 mM+SE BHB) sample sets. Plasma FA were determined by acid methylated and gas chromatography. The FA profile was multiplied by plasma FA concentration to approximate total concentration of each FA in the plasma. Data were analyzed using PROC MIXED (SAS 9.4) with a model containing the fixed effects of block, week (repeated measure), diagnosis, the interaction of diagnosis and week, and a random effect of cow. Results are expressed as mean ± SE. The mammary gland of ketotic cows took up a greater amount of FA than nonHYK cows (0.32 vs. 0.07 ± 0.4 mM; P < 0.01). The same was found for C16:0, C18:0, C18:1, and C18:2 (0.05 vs 0.01 ± 0.01, 0.05 vs. 0.01 ± 0.01, 0.07 vs. 0.01 ± 0.01, 0.11 vs. 0.02 ± 0.02 mM, for HYK and nonHYK, respectively; P < 0.01). However, the total FA disappearance (arterial-venous difference) relative to available concentration was not different (33.3 vs. 15.4 ± 9.6%; P = 0.19). This pattern was also observed for C16:0, C18:0, C18:1 and C18:2 (35.1 vs. 16.0 ± 9.9, 33.6 vs. 16.0 ± 9.6, 37.1 vs. 19.4 ± 9.5, 33.3 vs. 12.8 ± 9.9%, respectively; P > 0.17). Although no difference was observed in FTIR predicted milk FA output from the mammary gland, the proportion of FTIR predicted long chain FA and C18:1 relative to total milk fat was greater (P = 0.02) in HYK cows (31.6 vs. 27.5 ± 1.4% proportional C18:1). These data indicate that proportional uptake of FA by the mammary gland is not altered by HYK status. Cows with HYK do not have a different FTIR predicted FA profile, although an increase in relative C18:1 contribution may be detectable.