The objectives were to determine the optimal feeding amount of choline in a ruminally protected form to reduce the triacylglycerol (TAG) concentration in liver and to increase TAG in blood plasma of dairy cows. Pregnant, nonlactating multiparous Holstein cows (n = 77) were blocked by body condition score (3.59 ± 0.33) and assigned to treatment at 64 ± 10 d before calculated calving date. Dietary treatments were top-dressing of 0, 30, 60, 90, or 120 g/d of ruminally protected choline (RPC; Balchem Corp., New Hampton, NY) ions to supply the equivalent of 0, 6.5, 12.9, 19.4, and 25.8 g/d of choline ions. Diets were formulated to exceed nutrient requirements for maintenance and pregnancy and fed in ad libitum amounts for the first 5 d. From d 6 to 15, cows were restricted to consume approximately 31% of their net energy requirements to simulate early lactating cows in negative energy balance. Methionine intake was maintained throughout each 15-d period. Liver was biopsied at 5 and 14 d and analyzed for TAG and glycogen. Blood was sampled on d 5 and 14 and plasma analyzed for glucose, insulin, cholesterol, ß-hydroxybutyrate, long-chain fatty acids, and haptoglobin. On d 14, a mixture of saturated long-chain fatty acids, ground corn, and dried molasses (50:37:13) was offered (908 g, as-is basis) 10 h after the single daily feeding. Blood samples were collected for 19 h and plasma analyzed for TAG and cholesterol to assess apparent absorption of dietary fat. Mean dry matter intake and energy balance decreased from means of 9.5 to 3.3 kg/d and from 0.6 to -9.2 Mcal of net energy for lactation/d during the ad libitum and restricted feeding periods, respectively. Plasma concentrations of the lipid-soluble choline biomolecules, namely total phosphatidylcholines, total lysophosphatidylcholines, and sphingomyelin, increased with choline supplementation. Feed restriction increased plasma concentrations of ß-hydroxybutyrate and free long-chain fatty acids, whereas those of glucose, insulin, and total cholesterol decreased. During feed restriction, concentration of hepatic TAG and plasma haptoglobin decreased linearly, whereas concentration of hepatic glycogen tended to increase quadratically with increasing intake of RPC. After fat supplementation, mean plasma concentration of TAG increased by an average of 21% with intake of RPC ions, peaking at intakes of ≥6.5 g/d of RPC ion. In summary, feeding RPC ions to cows in negative energy balance had increasing lipotropic effects on the liver when consumed up to 25.8 g/d, whereas feeding only 6.5 g/d increased concentrations of hepatic glycogen and TAG in the blood.