Dairy cows may benefit from dietary supplementation with rumen-protected B vitamins and choline (RPBC) during the transition period. The objective was to determine the effect of a commercially available RPBC supplement on feed intake, milk yield, and gene expression and fat infiltration in the liver. Holstein dairy cows (n = 50) in the University of Guelph research herd were randomly assigned to receive 100 g/cow/d of RPBC (Jefo; choline, riboflavin, folic acid and B12 microencapsulated in a lipid matrix) or placebo (lipid supplement), fed in a common TMR from 3 wk before calving through 21 DIM. Cows were in freestall pens with individual feed bins (Insentec). DMI was measured daily and blood samples were collected weekly. Liver biopsies were taken at 4 and 14 ± 1 DIM to measure triacylglycerol (TAG) concentrations and expression of 28 genes selected to represent relevant aspects of liver metabolism. Data were analyzed with multivariable mixed linear regression models. All results are LS means ± SEM. DMI did not differ (P > 0.2) between RPBC and control (prepartum: 13.7 vs. 13.4 ± 0.5 kg/d; 0 to 21 DIM: 14.6 vs. 13.9 ± 0.70; 22 to 28 DIM: 17.9 vs. 16.9 ± 0.74). Mean blood BHB was lower at wk 3 in RPBC (0.6 vs 0.9 ± 0.12 mmol/L, P < 0.01) with no difference between treatments for mean blood concentrations of fatty acids (wk −1 or 1) and BHB at wk 1 or 2. Liver TAG was lower in primiparous cows at 4 DIM in RPBC (2.0 vs. 4.4 ± 1.2%, P = 0.05) but not at 14 DIM (2.2 vs. 3.2 ± 0.97%, P = 0.34) with no treatment effect for multiparous cows (4.6%, P > 0.75). Acyl-CoA oxidase 1, palmitoyl had lower mRNA abundance in RPBC with no difference between treatments for the other genes, but the expression of half of the genes assessed differed with DIM. Accounting for parity, DIM, fat and protein percentages, repeated test days, and a random effect of cow, cows that received RPBC tended to produce more milk across the first 3 DHIA tests (41.2 vs. 38.0 ± 1.4 kg/d, P = 0.11). The supplementation of RPBC may provide benefits to liver health and milk yield.