Rumen-protected choline (RPC) was evaluated for effects on the lipid and glycogen content of the liver and metabolic variables in the blood plasma of dairy cows. Thirty-two Holstein cows were allocated into two groups (RPC group with RPC supplementation and control group without RPC supplementation) 28 days before the expected calving. Cows were fed the experimental diet from 21 days before calving until day 60 of lactation. The diet of the RPC group was supplemented with 100 g/day of RPC from 21 days prepartum until calving and 200 g/day of RPC for 60 days postpartum, providing 25 and 50 g of choline, respectively. Liver samples were taken by percutaneous needle biopsy, then analysed for total lipid (TLl), triglyceride (TGl) and glycogen (GLYl) contents on days −21, +7, +35 and +60 relative to calving. Blood was collected on the same sampling days and 21 days after calving. Glucose, non-esterified fatty acid (NEFA), β-hydroxybutyrate (BHBA), triglyceride (TGp), total cholesterol (TCh), urea, ammonia and aspartate aminotransferase (AST) were determined from blood samples. The TLl and TGl contents were 25.0 ± 4.3 g and 25.3 ± 3.8 g per kg wet weight (mean ± SEM), respectively, lower in the RPC group than in the control animals. No significant differences were observed in the GLYl concentrations between the two groups. However, a lower TGl: GLYl ratio was shown in the liver of cows fed the RPC diet as compared to the controls. RPC supplementation decreased BHBA while increasing TGp concentrations were shown in the blood of cows fed the RPC diet, possibly as a consequence of improved lipoprotein synthesis in, and triglyceride excretion from, the liver, together with a reduced rate of ketogenesis.