During the peripartum period, dairy cows diagnosed with hyperketonemia experience both an increase in circulating FA, and a change in circulating FA profile, compared with non-hyperketonemic cows. Supplementation of dietary lipids can also result in subtle shifts in the circulating FA profile. As FA are known regulators of hepatic genes, these shifts could differentially influence gene expression. The objective was to determine the expression of gluconeogenic and oxidative genes in primary hepatocytes when exposed to an in vivo relevant FA profile and that profile enriched with additional C16:0, C18:0, or C18:1. Primary hepatocytes were isolated from 4 Holstein bull calves (<7 d) and cultured for 24 h. Treatments applied to cells for 24 h were no FA (1% BSA); 0.75 mM FA cocktail (3% C14:0, 27% C16:0, 23% C18:0, 31% C18:1, 8% C18:2, and 8% C18:3; to mimic the serum FA profile of dairy cattle at calving); 0.90 mM FA cocktail; 0.75 mM FA cocktail + 0.15 mM C16:0; 0.75 mM FA cocktail + 0.15 mM C18:0; and 0.75 FA cocktail mM + 0.15 mM C18:1. After harvest in TRIzol, samples were stored at −80°C until RNA extraction, purification, reverse transcription, and quantitative real time PCR. Expression of genes of interest (carnitine palmitoyltransferase 1A, pyruvate carboxylase, cytosolic and mitochondrial phosphoenolpyruvate carboxykinase [PEPCKc and PEPCKm], and glucose-6-phosphatase) was calculated relative to the geometric mean of 2 reference genes chosen by geNorm (ribosomal protein L32 and GAPDH). Data were analyzed using Proc Mixed (SAS 9.4) with the fixed effect of treatment and calf in the random statement. The addition of FA compared with no FA increased the expression of carnitine palmitoyl transferase 1A (2.22 vs. 3.96 ± 1.59 arbitrary units [AU]; P = 0.05) and PEPCKc (0.51 vs. 1.03 ± 0.28 AU; P = 0.03). Enrichment with individual FA did not affect the expression of the genes tested when compared with the 0.90 mM FA cocktail treatment (P ≥ 0.40). These results suggest additional shifts in circulating FA profile within a biological range have minimal additional effects on hepatic gluconeogenic and oxidative gene expression.