This experiment was conducted to investigate the effects of infusing milk fat precursors to external pudic artery on milk yield and uptake rule of short-chain fatty acids in mammary gland of dairy cows. Eight healthy Holstein cows were divided into two groups with four cows per group. A 2×2 cross-over experimental design was adopted. Cows in the two groups were fed diets with two kinds of roughages: corn stover (CS group) and alfalfa (MF group), and the diet concentrate to roughage ratio was 45∶55. The experiment was divided into two stages, and each stage lasted for 20 days, including 14 days of preliminary feeding period, 3 days of carrier infusion phase and 3 days of milk fat precursors infusion phase. During carrier infusion period, MF group and CS group received carrier infusion, and were named MFC₁ group and CSC group, respectively; during milk fat precursors infusion period, MF group continued to accept carrier infusion (MFC₂ group), while CS group accepted milk fat precursors infusion (CSF group). Milk yield, dry matter intake and milk composition were determined. Plasma in caudal artery and internal mammary vein was collected to determine short-chain fatty acid concentrations and fatty acid proportions. The results showed as follows: 1) before milk fat precursors infusion, CSC group had significantly lower milk yield, 4% fat corrected milk (FCM) yield, milk fat yield, milk protein yield and milk fat producing efficiency than MFC1 group (P<0.05), and short-chain fatty acid concentration and short-chain fatty acid to long-chain fatty acid rate in plasma of caudal artery in CSC group tended to be higher than those in MFC₁ group (0.05≤P < 0.10). 2) After milk fat precursors infusion, milk fat producing efficiency and milk protein percentage in CSF group were significantly higher than those in CSC group (P<0.05), and CSF group still below in MFC₂ group in milk yield, 4% FCM yield, milk fat percentage and milk protein percentage, but no significant differences was found between groups (P>0.05); CSF group was significantly higher than MFC₂ group in milk fat producing efficiency (P<0.05). 3) After milk fat precursors infusion, the concentrations of acetic acid, propionic acid and short-chain fatty acids in plasma of caudal artery in CSF group were significantly higher than those in CSC group (P<0.05), and the concentrations of acetic acid, butyric acid and short-chain fatty acids in CSF group was significantly higher than those in MFC₂ group (P<0.05), but short-chain fatty acids to long-chain fatty acids rate in CSC group tended to be lower than that in MFC₂ group (0.05≤P < 0.10). 4) Except that artery-vein difference of butyric acid in CSF group tended to be higher than that in CSC group (0.05≤P < 0.10), and there were no significant differences in uptake quantity, uptake rate and artery-vein difference of acetic acid, propionic acid, butyric acid and short-chain fatty acids between CSC group and MFC₁ group, CSC group and CSF group, CSF group and MFC₂ group (P>0.05). The results indicate that dairy cows fed corn stover as roughage infused milk fat precursors can significantly increase the milk yield, milk fat percentage and milk fat yield, and closely achieves the levels in dairy cows fed alfalfa as roughage, especially has producing efficiency of milk fat, but has no significant effects on uptake quantity, uptake rate of short-chain fatty acids in mammary gland.