Dietary fatty acids (FA) and one-carbon donors may influence ceramide and phosphatidylcholine (PC) synthesis in cows with implications for health. In a 5 × 5 Latin square design, 5 rumen-cannulated Holstein cows (214 ± 4.9 DIM; 3.2 ± 1.1 parity) were enrolled in a study with each experimental period lasting 6 d. Abomasal infusates consisted of (1) palmitic acid (PA; 98% 16:0 of total fat), (2) PA + choline chloride (PA+C; 50 g/d choline ion), (3) PA + L-serine (PA+S; 170 g/d L-serine), (4) behenic acid (BA; 92% 22:0 of total fat), and (5) a docosahexaenoic acid algal oil (47.5% DHA of total fat). Emulsions were formulated to provide 301 g/d of total FA and were balanced to provide a minimum of 40 and 19 g/d of 16:0 and glycerol, respectively. Cows were fed a corn silage-based diet. Blood was collected on d 0, 1, 3, 5, and 6. Liver was biopsied on d 6. Plasma choline metabolites, and hepatic ceramides and PC were measured using mass spec. For univariates, ANOVA was performed including fixed effects of treatment and time, and their interaction. Generalized log-transformed lipidomic data were analyzed using multivariate ANOVA. Although treatment did not modify milk yield, milk fat yield and content were lowest for DHA-infused cows (P < 0.05). Plasma triglyceride, cholesterol, and insulin levels were lowest with DHA infusion (P < 0.05). Plasma choline, betaine, dimethylglycine, and trimethylamine N-oxide levels were greatest in cows infused PA+C (P < 0.01). Hepatic ceramide-d18:2/22:0 and -d16:1/22:0 levels were elevated in cows infused BA (P < 0.05). Infusion of DHA increased hepatic PC containing 4 or more double bonds (52 out of 61 PC; e.g., PC-14:0/22:6 or 22:0/20:4; P < 0.05), relative to other treatments. Hepatic PC containing saturated FA were lowest for DHA-infused cows. While PA+C increased hepatic PC with moderate saturation (P < 0.05), PA+C decreased highly unsaturated PC when compared with PA (P < 0.05). A similar outcome was observed for cows infused PA+S. Abomasal infusion of saturated FA, DHA, and one-carbon donors uniquely modifies hepatic ceramide and PC levels in lactating cows.