Cobalamin (vitamin B12, VB12) has the potential to enhance the digestion of dietary nutrients, optimize energy distribution, and stimulate the proliferation of mammary epithelial cells (MECs), which in turn can promote milk production performance. The influence of VB12 on nutrient digestion and energy distribution has been studied; however, its effects on MEC proliferation and the underlying mechanism remain unclear. This study investigated the influence of coated VB12 (CVB12) on milk production, nutrient digestion, MECs proliferation and milk fatty acid synthesis in dairy cows. This in vivo study involved 88 multiparous Holstein cows with similar body weight (BW; 633 ± 16.2 kg), previous 305-d milk production (9035 ± 186.2 kg), parity (3.1 ± 0.28), and age (5.6 ± 0.31 years). These cows were selected from 6 weeks preparturition to 9 weeks postparturition. They were blocked by parity, previous milk yield, and expected calving date and divided into four treatment groups of 22 cows per group: control, low CVB12 (LCVB12), medium CVB12 (MCVB12), and high CVB12 (HCVB12), receiving 0, 6, 12, and 18 mg of VB12 per cow per day, respectively. Although dry matter intake was unaffected, yields of actual milk, 4% fat-corrected milk, energy-corrected milk, and milk components (fat, true protein, and lactose) increased quadratically (P < 0.05). Milk fat content increased linearly (P = 0.021) with no significant changes in milk true protein and lactose contents after CVB12 supplementation (P > 0.05). The apparent nutrient digestibility of dry matter, organic matter, crude protein, ether extract, neutral detergent fiber, and acid detergent fiber increased linearly (P < 0.05) following the provision of CVB12. Serum contents of VB12, glucose, and total protein linearly increased (P < 0.05), while those of methylmalonic acid, nonesterified fatty acid, and β-hydroxybutyric acid linearly decreased (P < 0.05) following CVB12 provision. Supplementing 12 mg/d of CVB12 promoted MECs proliferation and fatty acid synthesis, as indicated by the upregulated expression of proliferation-related proteins, the suppression of apoptosis-related proteins, the phosphorylation of protein kinase B (Akt) and rapamycin target protein (mTOR), and the increased expression of fatty acid synthesis-related proteins. The in vitro study verified the promoting effect of VB12 on MECs proliferation and further elucidated that VB12 regulates MECs proliferation in dairy cows through the Akt/mTOR signaling pathway. In conclusion, CVB12 increases milk and milk component production by promoting nutrient digestion, energy distribution, and MECs proliferation through the Akt/mTOR signaling pathway, and fatty acid synthesis-related proteins expression. These findings offer a robust theoretical foundation for future strategies aimed at enhancing milk performance in dairy cows through CVB12 nutrition.