Dietary fatty acids (FA) influence the composition of sphingolipids and glycerophospholipids, and modulate energy metabolism. 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. Skeletal muscle was biopsied on d 6. Plasma metabolites and muscle lipids were measured using untargeted metabolomics. Generalized log-transformed data were analyzed using ANOVA (effects of treatment, time, and their interaction). Plasma FA (e.g., DHA and arachidonic acid) increased in response to DHA infusion (P < 0.05). Infusing DHA reduced plasma tyrosine, leucine, alanine, and pyruvic, citric, and uric acids, and increased plasma succinic acid (P < 0.05). Plasma choline and betaine increased in PA+C, and serine increased in PA+S (P < 0.05). Specific DHA-containing plasma triacylglycerols (TAG, n = 44) and PC (n = 8) were only detected in DHA-infused cows. Discriminant analysis revealed a unique lipidomic signature in response to DHA, with most of the treatment differences reflected as changes in plasma PC (111/143; P < 0.05). Saturated PC increased in PA (e.g., PC-16:0/20:4; P < 0.05), whereas DHA increased very-long chain PC and TAG containing DHA (e.g., PC-40:4 and TAG-16:0/16:0/22:6, respectively; P < 0.05). Distinctive treatment effects on muscle lipids were observed in discriminant analysis, characterized by changes in PC, TAG and ceramides (e.g., ceramide-d18:1/22:0 was higher in BA, relative to PA and DHA; P < 0.05). We conclude that the abomasal infusion of FA and one-carbon donors modifies the plasma and muscle metabolome.