To investigate the effects of a dietary pantothenic acid (PA) supplementation on duodenal nutrient flows, blood and milk variables and especially on duodenal PA flows and PA concentrations in blood and milk German Holstein cows, equipped with cannulas in the dorsal sac of the rumen and in the proximal duodenum were used. In the first experiment of the study two dry and six lactating cows received a diet with a forage to concentrate (F:C) ratio of 34:66 (high concentrate, HC), whereas in the second experiment a diet with a F:C ratio of 66:34 (high forage, HF) was fed to four dry and five lactating cows. The cows received both rations with or without 1 g PA/day. By supplementing PA to the HC ration, the molar percentage of acetic acid increased, whereas the concentration of total short chain fatty acids, the efficiency of microbial protein synthesis in the rumen and the serum glucose levels, decreased. With the HF ration the PA decreased the molar percentage of propionic acid and increased the amount of ruminally fermented organic matter. Furthermore, PA supplementation only increased the duodenal PA flow with the HF ration, while the serum and milk PA concentrations and milk PA yields were not affected. Based on the results of the present study, a rumen-unprotected supplementation of PA makes no sense.