Ceramide is synthesized by the condensation of palmitoyl-CoA with serine in a reaction controlled by serine palmitoyltransferase (SPT). The enhanced hepatocyte uptake of saturated fatty acids contributes to the activation of SPT and de novo ceramide synthesis. In dairy cattle, hepatic ceramide accrual has been linked to metabolic disease, and palmitic acid feeding increases hepatic and circulating ceramide. Therefore, we hypothesized that palmitic acid increases hepatocyte ceramide concentrations by activating SPT. To test our hypothesis, primary hepatocytes isolated from 4 neonatal Holstein calves were maintained as monolayer cultures for 24 h. At 24 h, media was refreshed with 1% fatty acid-free BSA, and cells were unsupplemented (control) or exposed to 1 mM palmitic acid in the absence or presence of 10 µM myriocin (SPT inhibitor) for 18 h before collection in PBS. Ceramide, monohexosylceramides (GlcCer), and lactosylceramides (LacCer) were extracted then quantified using mass spectrometry. For normalization purposes, cellular protein was quantified using the bicinchoninic acid assay. Data were analyzed using a mixed model (fixed effect of treatment and random effect of calf). Reflective of bovine liver and plasma, C24:0, C22:0, and C16:0 ceramide represented 30, 21, and 20% of total ceramide in unsupplemented cells, respectively. A similar profile was observed for GlcCer, whereas C16:0 LacCer represented the most abundant LacCer. Palmitic acid tended to increase total ceramide and GlcCer by 53 and 63%, respectively (P ≤ 0.10), relative to control. Specifically, palmitic acid significantly increased C16:0, C18:0, and C22:0 ceramide (P < 0.05), and tended to increase C24:0 ceramide (P = 0.07). Moreover, palmitic acid increased C16:0 and C16:1 GlcCer (P < 0.01). Notably, the inhibition of SPT by myriocin prevented palmitic acid-induced de novo ceramide synthesis. For example, myriocin completely prevented the ability of palmitic acid to increase C16:0 and C24:0 ceramide (P < 0.05). We conclude that palmitic acid induces de novo ceramide synthesis in bovine primary hepatocytes.