Adipose tissue mobilization increases circulating fatty acid (FA) concentration, hepatic FA uptake, and influences hepatic metabolism. The objective of this experiment was to examine the effect of FA challenge on complete and incomplete oxidation, glucose output, and oxidative stress in bovine primary hepatocytes. Primary hepatocytes isolated from 3 neonatal Holstein calves were maintained as monolayer cultures for 24 h. At 24 h, media was refreshed with a glucose-free media containing only pyruvate as a gluconeogenic precursor, and cells were exposed to 0 or a 1 mM FA cocktail that reflected the circulating FA profile at calving. After 21h of treatment, ¹⁴C-labeled palmitate or pyruvate was added to the media and both CO₂ and acid soluble products (ASP) were collected after a 3-h incubation. Media was harvested to quantify glucose and reactive oxygen species (ROS). Cell lysates were collected and DNA quantified to normalize all data. Data were analyzed by PROC MIXED (SAS 9.4) in a model accounting for fixed effect of FA treatment and random effect of calf. Data are reported as least squares means ± SE and differences declared at P ≤ 0.10 and tendencies at P < 0.15. Fatty acid treatment decreased (P = 0.01) the relative recovery of pyruvate label as CO₂ (3.8 vs. 1.94 ± 0.31 arbitrary units) and increased (P = 0.02) label recovery as ASP (20.0 vs. 27.3 ± 1.4 arbitrary units). Recovery of palmitate label as CO₂ tended to be greater (P = 0.14) in FA treated cells (0.87 vs. 1.05 ± 0.15 arbitrary units) but ASP from palmitate was not affected (P > 0.18) by FA treatment. Glucose output from cells exposed to FA was increased (P < 0.007) by 35%. Treatment with FA increased (P = 0.07) ROS in cell culture media. Fatty acid challenge appears to shift oxidative and gluconeogenic capacity in a substrate-specific manner. Decreased complete and increased incomplete oxidation of pyruvate indicates a shift of pyruvate conversion toward ASP or glucose production. Conversely, fatty acid challenge increased complete oxidation of palmitate, consistent with previous indications that FA can upregulate key oxidative enzymes at the time of calving.