We previously reported that supplementation of rumen-protected choline (RPC) reduces the hepatic triacylglycerol concentration in periparturient dairy cows during early lactation. Here, we investigated the effect of RPC on the transcript levels of lipid metabolism-related genes in liver and adipose tissue biopsies, taken at wk -3, 1, 3, and 6 after calving, to elucidate the mechanisms underlying this RPC-induced reduction of hepatic lipidosis. Sixteen multiparous cows were blocked into 8 pairs and randomly allocated to either 1 of 2 treatments, with or without RPC. Treatments were applied from 3 wk before to 6 wk after calving. Both groups received a basal diet and concentrate mixture. One group received RPC supplementation, resulting in an intake of 14.4 g of choline per day, whereas controls received an isoenergetic mixture of palm oil and additional soybean meal. Liver and adipose tissue biopsies were taken at wk -3, 1, 3, and 6 to determine the mRNA abundance of 18 key genes involved in liver and adipose tissue lipid and energy metabolism. Milk samples were collected in wk 1, 2, 3, and 6 postpartum for analysis of milk fatty acid (FA) composition. The RPC-induced reduction in hepatic lipidosis could not be attributed to altered lipolysis in adipose tissue, as no treatment effect was observed on the expression of peroxisome proliferator-activated receptor ¿, lipoprotein lipase, or FA synthase in adipose tissue, or on the milk FA composition. Rumen-protected choline supplementation increased the expression of FA transport protein 5 and carnitine transporter SLC22A5 in the liver, suggesting an increase in the capacity of FA uptake and intracellular transport, but no treatment effect was observed on carnitine palmitoyl transferase 1A, transporting long-chain FA into mitochondria. In the same organ, RPC appeared to promote apolipoprotein B-containing lipoprotein assembly, as shown by elevated microsomal triglyceride transfer protein expression and apolipoprotein B100 expression. Cows supplemented with RPC displayed elevated levels of glucose transporter 2 mRNA and a reduced peak in pyruvate carboxylase mRNA immediately after calving, showing that supplementation also resulted in improved carbohydrate metabolism. The results from this study suggest that RPC supplementation reduces liver triacylglycerol by improved FA processing and very-low-density lipoprotein synthesis, and RPC also benefits hepatic carbohydrate metabolism.