Supplementation of rumen-protected choline (RPC; ReaShure, Balchem Corp., New Hampton, NY) during late-pregnancy in Holstein cows improves development of the offspring’s immune system and growth. Here we evaluated if RPC concurrently altered the systemic metabolome. Twenty-four Holstein heifers born to cows fed a basal diet [1.59 Mcal/kg DM, 15.8% CP, 2.9% methionine (% MP) and a lysine to methionine ratio of 2.6] without (control) or with RPC (last 21 d of gestation at a rate of 60 g/d) were used. Immediately after birth whole blood samples were taken and stored at −20°C. Global metabolomics profiling was performed on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18-pfp 100 × 2.1 mm, 2-μm column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. This is a polar embedded stationary phase that provides comprehensive coverage, but does have some limitation is the coverage of very polar species. The flow rate was 350 μL/min with a column temperature of 25°C. A total of 7745 molecular features were detected of which 356 peaks with putative identification represent 305 unique metabolites, including amino acids, benzoic acids, lipid molecules, carbohydrates, purines, pyrimidines, vitamins, and other intermediate and secondary metabolites. Statistical analysis was performed using the Mixed procedure of SAS for molecular features with putative identification. There were 12 unique metabolites altered by RPC (P < 0.05). In particular glycodeoxycholic acid, lactate, 5′deoxyadenosine, and l-arginine were reduced by RPC, whereas cytidine, α-linolenic acid, cytosine, 1-aminocyclopropane-1-carboxylate, glutathione disulfide, pipecolate, threonine/homoserine, and l-proline increased by RPC. Further data analyses are needed to unravel the specific consequences of these changes. Overall, maternal supplementation with RPC during late-gestation changes the offspring’s systemic metabolome, which is likely to be involved in the positive effect of prenatal RPC supplementation on neonatal heifer growth and immune responses.