Simple SummaryVitamin B12is produced by ruminal microbes, and dietary Co is essential in this process, ultimately residing at the center of the corrin ring of the vitamin. Together with folate, vitamin B12participates in the remethylation of homocysteine to generate methionine. Several studies with dairy cows have highlighted the positive effect of supplementing Co above current estimated requirements on the ruminal synthesis of vitamin B12. Studies with dairy cows have yielded inconsistent results in terms of lactation performance both in early- and mid-lactation phases. Despite this, it was clearly demonstrated that concentrations of Co and vitamin B12in the liver rise markedly after calving when periparturient Holstein cows are fed supplemental Co. We sought to assess if Co source, folic acid, and rumen-protected methionine (RPM) fed in various combinations around parturition have an impact on performance and one-carbon metabolism in the liver. Despite a lack of effect on performance, feeding ruminally available folic acid with an experimental source of Co and RPM had the strongest effect on the activity of hepatic methionine adenosyltransferase, which produces the methyl donor S-adenosylmethionine. Thus, these nutrients may impact methyl-group-requiring reactions in the liver during the transition period.AbstractVitamin B12plays a role in the remethylation of homocysteine to Met, which then serves as a substrate for Met adenosyltransferase (MAT) to synthesize S-adenosylmethionine (SAM). We investigated effects of feeding two cobalt sources [Co-glucoheptonate (CoPro) or CoPectin, Zinpro Corp.], an experimental ruminally-available source of folic acid (FOA), and rumen-protected Met (RPM) on performance and hepatic one-carbon metabolism in peripartal Holstein cows. From −30 to 30 d around calving, 72 multiparous cows were randomly allocated to: CoPro, CoPro + FOA, CoPectin + FOA, or CoPectin + FOA + RPM. The Co treatments delivered 1 mg Co/kg of DM (CoPro or CoPectin), each FOA group received 50 mg/d FOA, and RPM was fed at 0.09% of DM intake (DMI). Milk yield and DMI were not affected. Compared with other groups, the percentage of milk protein was greater after the second week of lactation in CoPectin + FOA + RPM. Compared with CoPro or CoPro + FOA, feeding CoPectin + FOA or CoPectin + FOA + RPM led to a greater activity of MAT at 7 to 15 d postcalving. For betaine–homocysteine S-methyltransferase, CoPro together with CoPectin + FOA + RPM cows had greater activity at 7 and 15 d than CoPro + FOA. Overall, supplying FOA with CoPectin or CoPectin plus RPM may enhance S-adenosylmethionine synthesis via MAT in the liver after parturition. As such, these nutrients may impact methylation reactions and liver function.