Supplementation of rumen-protected choline (RPC; ReaShure, Balchem Corp., New Hampton, NY) reduced liver fat accumulation in feed restricted pregnant, nonlactating dairy cows. In these same cows, we evaluated if RPC concurrently altered hepatic gene expression. Pregnant, nonlactating multiparous Holstein cows (n = 77) were enrolled at 64 ± 10 d before expected calving date. Dietary treatments were 0, 30, 60, 90, and 120 g/d of ReaShure. Experimental periods were 14 d with 5 d of ad libitum intake (AL), and 9 d of feed restriction to consume 31% of caloric requirement (FR) to induce negative nutrient balance. Methionine was supplemented during the FR period to maintain the same daily intake of the AL period. Liver tissue was collected on d 5 and 14. Transcript abundance was determined by RT-qPCR using the Fluidigm assay for 93 target genes involved in assembly and secretion of VLDL, CDP-pathway, gluconeogenesis, inflammation, oxidative stress, transcription factors, metabolism of lipid, lipoprotein, and phosphatidylcholine; and 3 reference genes. The ΔCt values were calculated relative to the geometric mean of the reference genes and used for statistical analyses using the Mixed procedure of SAS. Birth weight of the calves and number of days prepartum at enrollment were used as covariates. Expression of genes was affected (P < 0.05) by RPC during both periods. After cows developed fatty liver (d14) there were 20 differentially expressed genes (DEG), whereas there wer 13 DEG on d5. Changes in expression of genes due to RPC suggest increase in phosphatidylcholine metabolism, and reduction of TAG synthesis, among others. In conclusion, RPC regulates expression of genes in the liver, more extensively under fatty liver conditions to reduce TAG contents. Results demonstrate that regulation of hepatic gene expression is one mechanism by which RPC supplementation leads to the optimal performance of transition cows.