This experiment was conducted to study the effects of infusing amino acid (AA) mixture into external pubic artery on lactating performance, short chain fatty acid (SCFA) concentrations and their proportions in caudal artery and mammary vein blood, and the uptake rule of SCFA in mammary gland of mid-lactation Holstein cows under corn straw diet condition using arteriovenous blood intubation technique. The experiment used a 2×2 cross experiment design. Eight healthy multifarious (2 to 3 parities) Holstein cows with good body condition, similar body weight and the milk yield of (20.17±1.28) kg/d were randomly assigned in 2 groups[alfalfa group (MF group) and corn straw group (CS group)] which were fed different diets with 4 cows of each group. In MF group and CS group, the dietary forage-to-concentrate ratio both was 45:55 and the concentrate composition was the same, but the forage composition was different. The forage of MF group was composed of alfalfa hay, corn silage and Chinese wildrye, and in the CS group, the corn straw was used to replace the total forage of MF group diet. The experiment was divided into 2 stages, and each stage lasted for 20 days, including the diet adjustment period (pre feeding period) of 14 days, carrier infusion phase of 3 days and experimental infusion phase of 3 days. In the first stage, dairy cows of MF group (positive control group 1) and CS group (control group) were infused the carrier during carrier infusion phase. During experimental infusion phase, the dairy cows of MF group were infused carrier continuously (positive control group 2), and the dairy cows of CS group were infused AA mixture. In the second phase, the dairy cows of CS group and MF group were swapped, and the processing method was the same as the first stage. Blood and milk samples were taken from dairy cows on the last 2 days of infusion in each experimental infusion phase. The results showed as follows:infusing AA mixture into external pubic artery of dairy cows in CS group significantly improved milk protein rate (P<0.05), and milk yield, fat corrected milk (FCM) yield, milk fat rate, milk fat yield and milk protein yield were promoted, but some indices of them still significantly lower than MF group (P<0.05). Infusing AA mixture into external pubic artery of dairy cows in CS group had a tendency to increase the concentration of acetic acid in mammary vein blood (P=0.09), and significantly reduced the arteriovenous difference of acetic acid and the gaps of the uptake quantity and uptake efficiency of acetic acid in the mammary gland between CS group and MF group. Infusing AA mixture into external pubic artery of dairy cows in CS group had a certain promoting effect on increasing acetic acid/propionic acid and (acetic acid+butyric acid)/propionic acid in caudal artery blood (P>0.05). In conclusion, infusing AA mixture into external pubic artery of dairy cows using corn straw as forage can significantly improve milk protein rate, increase the concentration of acetic acid in internal mammary vein blood, and reduce the gaps of intake quantity and intake efficiency of acetic acid in mammary gland with the dairy cows using alfalfa hay, corn silage and Chinese wildrye as forage.