Enhanced post-ruminal supply of choline (CHL) may increase flux through the methionine (Met) cycle to improve immunometabolic status during a negative nutrient balance (NNB). The objective was to investigate effects of post-ruminal CHL supply during a feed restriction-induced negative NNB on: 1) hepatic activity of cystathionine β-synthase (CBS) and methionine synthase (MTR) and expression of enzymes and metabolite concentrations in the transsulfuration pathway and Met cycle and 2) plasma biomarkers of liver function, inflammation and oxidative stress. Ten primiparous rumen-cannulated Holstein cows (158 ± 24 DIM) were used in a replicated 5 × 5 Latin square design with 4-d treatment periods and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water (A0), restricted intake (R; 60% of NE_L requirements) with abomasal infusion of water (R0) or R plus abomasal infusion of 6.25, 12.5, or 25g/d CHL ion. Liver tissue was collected on d 5 when infusions ended for metabolomics and radioactive enzyme assays, and blood on d 1, 3 and 5 for measurement of plasma biomarkers via commercial kits. Statistical contrasts were A0 vs. R0, R vs. the average of CHL doses and tests of linear and quadratic effects. While R decreased MTR activity (P = 0.02), it tended to increase linearly with CHL (P = 0.07). Activity of CBS tended to be lower with R (P = 0.07) and decreased linearly with CHL (P = 0.03). Hepatic glutathione content was not different with R or CHL (P > 0.10) but taurine tended to be greater with CHL (P = 0.09). Betaine and carnitine were greater with R (P < 0.05) and further increased with CHL (P < 0.05). Plasma aspartate aminotransferase and bilirubin increased with R (P < 0.05) but decreased with CHL (P < 0.05). Data suggest that enhanced supply of CHL during NNB decreases entry of homocysteine to the transsulfuration pathway, potentially favoring remethylation to Met by acquiring a methyl group from betaine. As such, Met may provide methyl groups for synthesis of carnitine. Changes in blood biomarkers suggest a beneficial effect of CHL on liver function during NNB.