This experiment was conducted to investigate the effects of dietary cobalamin on lactation perform⁃ ance, rumen fermentation and liver gluconogenesis of Holstein dairy cows. Sixty Holstein dairy cows with simi⁃ lar body weight of (652.0±42.5) kg, parity of (2.04±0.39), lactation days of (104.7±31.2) and average milk yield of (33.6±18.7) kg / d were divided into 4 groups (15 cows per group) according to random block design, and the cobalamin was supplemented with 0 (control group), 6 (LCCA group), 12 (MCCA group) and 18 mg / d (HCCA group) in the form of coated cobalamin (CCA), respectively. The pre⁃trial period las⁃ ted for 20 days and the experimental period lasted for 60 days. The results showed as follows, with the increase of CCA supplemental level: 1) there was no significant difference in the body weight change of dairy cows (P>0.05), the dry matter intake was linearly decreased (P<0.05), and the yields of 4% fat correction milk and milk components (milk fat, milk protein and lactose) and feed efficiency were linearly increased (P < 0.05); 2) the pH in rumen had no significant change (P>0.05), the total volatile fatty acid content and pro⁃ pionate molar percentage in rumen were linearly increased (P<0.05), and the acetate molar percentage and ac⁃ etate to propionate ratio in rumen were linearly decreased (P<0.05); 3) the numbers of Ruminobacter amylo⁃ philus, Prevotella ruminicola, Butyrivibrio fibrisolvens, Ruminococcus albus, Ruminococcus flavefaciens, to⁃ tal fungi and total protozoa in rumen were increased linearly (P<0.05), while the numbers of Fibrobacter suc⁃ cinogenes and total bacteria in rumen were quadraticly changed (P<0.05); 4) the contents of glucose, total protein, albumin, insulin and cobalamin in serum were linearly increased (P<0.05), while the contents of u⁃ rea nitrogen, triglyceride, non⁃esterification fatty acid and β⁃hydroxybutyrate in serum had no significant chan⁃ ges (P > 0. 05); 5) the activities of methylmalonyl⁃coenzyme A mutase, pyruvate carboxylase and phos⁃ phoenolpyruvate carboxykinase and the cobalamin content in liver were linearly increased (P<0.05), while the activities of glucose⁃6⁃phosphatase, fructose⁃1,6⁃bisphosphatase and glycogen content in liver had no signifi⁃ cant changes (P >0.05). In conclusion, dietary CCA can improve lactation performance, rumen propionate content and liver gluconogenic key enzyme activities of dairy cows, and it is recommended that the supplemen⁃ tal level of cobalamin in the form of CCA is 12 mg / d in dairy cow diets.[ Chinese Journal of Animal Nutri⁃ tion, 2024, 36(2):1020-1028]