Insufficient peripartal supply of methionine (Met) and choline (Chol) could compromise hepatic metabolism in dairy cows. The objectives of this study were to isolate primary dairy cow hepatocytes to examine transmethylation, transsulfuration, and CDP-choline pathways in response to Met or Chol. Hepatocytes were isolated from mid-lactation multiparous Holstein cows from liver biopsy and maintained in monolayer culture. Purity of isolated hepatocytes were determined using flow cytometry with cytokeratin 18 as marker for hepatocytes. Hepatocytes were treated with control (Met and Chol free), Met (40 μM) or Chol (80 mg/dL) for 24 h in triplicates before harvested for gene and protein abundance analyses using qPCR and Western blot. Data were analyzed with PROC MIXED of SAS 9.4 with fixed effect of treatment and random effect of wells. Flow cytometry assay revealed 91% of isolated hepatocytes are cytokeratin 18 positive. Compared with control and Chol, MAT1A and BHMT protein abundance were greater (P < 0.05) in Met treated hepatocytes. Similarly, mRNA and abundance of genes in transmethylation pathway (MAT1A, PEMT, SAHH, BHMT, SARDH) were greater (P < 0.05) in Met treated hepatocytes, suggesting enhanced transmethylation and phosphatidylcholine (PC) synthesis in resonse to Met supply. The abundance of genes associated with the CDP-choline pathway (CHKA, CHKB, PCYT1a, and CEPT1) were greater (P < 0.05) in hepatocytes treated with Chol compared with control or Met, suggesting enhanced PC synthesis via CDP-choline pathway. Protein abundance of cystathionine β-synthase (CBS) was greater (P < 0.05) in hepatocytes treated with Met compared with control or Chol. Additionally, mRNA abundance of genes in transsulfuration pathway (CBS, CSAD, GCLC, and GSR) were also greater (P < 0.05) in hepatocytes treated with Met compared with control or Chol, indicating enhanced transsulfuration pathway in response to Met supply. Overall, these findings suggest transmethylation and transsulfuration appear more responsive to Met supply, while the CDP-choline pathway is more responsive to Chol supply.