|Abstract:||The onset of lactation in dairy cows is characterized by high output of methylated compounds in milk when sources of methyl group are in short supply. Methionine (MET) and choline (CHOL) are key methyl donors and their availability during this time may be limiting for milk production, hepatic lipid metabolism, and immune function. Supplementing rumen-protected MET and CHOL may improve overall performance and health of transition cows. Physiological and molecular mechanisms for methyl donor supplementation benefits are unknown. Objectives were to evaluate performance, immunometabolic status, liver function, and the underlying mechanisms in response to MET and CHOL supplementation. Eighty-one multiparous Holstein cows were used in a randomized complete block design with 2×2 factorial arrangement of MET (Smartamine M, Adisseo NA) and CHOL (ReaShure, Balchem Inc.) level (with or without). Treatments were control (CON), no MET or CHO; CON+MET (SMA); CON+CHO (REA); and CON+MET+CHO (MIX). From −50 d to −21 d before expected calving, all cows received the same diet (1.40 Mcal/kg DM). From −21 d to calving, cows received the same close up diet (1.52 Mcal/kg DM) and were assigned randomly to treatments (CON, SMA, REA, or MIX). From calving to 30 DIM, cows were on the same postpartal diet (1.71 Mcal/kg DM) and continued to receive the same treatments through 30 DIM. MET supplementation was adjusted daily at 0.08% DM of diet and CHOL was supplemented at 60 g/cow/d. Blood (-10, 4, 8, 20, and 30 d) and liver (-10, 7, 21, and 30 d) samples were harvested for biomarker and molecular analyses. Incidence of clinical ketosis and retained placenta tended to be lower in MET-supplemented cows. MET supplementation led to greater DMI during close-up (P = 0.01) and first 30 d postpartum (P = 0.02). Milk yield (P = 0.03) and milk protein % (P < 0.01) also were greater in MET- compared with CHOL-supplemented cows. The greater overall plasma albumin concentration (P = 0.04), blood neutrophil phagocytosis capacity (P = 0.01), and neutrophil oxidative burst (P = 0.03) in MET-supplemented cows underscored a better liver function and immune status. In addition, the greater concentrations of hepatic reduced and total (P = 0.01) glutathione indicated a lower degree of oxidative stress in cows fed MET compared with CHOL. MET-supplemented cows had greater (0.38 vs. 0.27; SEM = 0.05; P = 0.02) methionine adenosyltransferase 1A (MAT1A) and phosphatidylethanolamine methyltransferase (PEMT) expression (0.74 vs. 0.58; SEM = 0.08; P = 0.05). Greater (0.93 vs. 0.74; SEM = 0.05; P = 0.01) S-adenosylhomocysteine hydrolase (SAHH) and CBS (1.16 vs. 1.02; SEM = 0.07; P = 0.04) as well as lower (23.4 vs. 29.7 nmol product h-1 mg protein-1; SEM = 2.9; P = 0.04) MTR activity were also detected in MET but not CHOL-supplemented cows. Although greater (1.07 vs. 0.93; SEM = 0.05; P = 0.01) expression of betaine aldehyde dehydrogenase (BADH) was observed in response to CHOL supplementation, expression of (BHMT) and MTR and BHMT enzyme activity did not change (P > 0.05). Furthermore, overall MTR enzyme activity was lower (23.5 vs. 29.6 nmol product h-1 mg protein-1; SEM = 2.9; P = 0.05) in CHOL cows. In terms of AA profile, MET supplementation led to greater (P < 0.01) circulating methionine and proportion of methionine in the essential AA pool, total AA, and total sulfur-containing compounds. Lysine in total AA also tends to be greater (P = 0.08) in these cows, indicating a better overall AA profile. Sulfur-containing compounds (cystathionine, cystine, homocystine, and taurine) also were greater (P < 0.05) in MET-supplemented cows, indicating an enriched sulfur-containing compound pool due to enhanced transsulfuration activity. Circulating essential AA (P = 0.06) and total AA (P = 0.03) concentrations were greater in MET cows due to greater lysine, arginine, tryptophan, threonine, proline, asparagine, alanine, and citrulline. In contrast, tryptophan and cystine were greater. Plasma 3-methylhistidine concentration was lower (P = 0.02) in response to CHOL, suggesting less tissue protein mobilization in these cows. Overall, results indicate that MET supplementation improves performance of transition cows through a combination of better immunometabolic status, plasma AA profile, and a reduction in oxidative stress; insufficient regeneration of MET may be one reason for the lack of effect with supplemental CHOL.