Hyperlipidemia develops with hepatic steatosis and insulin antagonism in domestic ruminants. Enhanced hepatic fatty acid uptake contributes to de novo ceramide synthesis in non-ruminants. In turn, circulating ceramide antagonizes insulin sensitivity in skeletal muscle tissue. Our objective was to determine whether the induction of hyperlipidemia modifies hepatic ceramide concentrations and the mRNA expression of ceramide synthases (CerS). Six non-pregnant, nonlactating Holstein dairy cows (682 kg ± 22), were used in a crossover design with treatments consisting of i.v. infusion (100 mL/h) of either saline (control) or triacylglycerol (TG) emulsion (Intralipid 20%; Frasenius Kabi) for 16 consecutive hours. The feeding level was set at 120% of estimated energy requirement with meals offered every 2 h. Blood was collected at regular intervals and liver biopsied at 16 h. LC/MS was used to quantify ceramides, monohexosylceramides (GlcCer), and lactosylceramides (LacCer) in liver. Real-time PCR was utilized to evaluate CerS isoform expression. Data were analyzed using a mixed model (fixed effects of treatment and random effect of cow). As previously established, TG infusion increased hepatic TG, and plasma total fatty acid and ceramide levels by 16 h, relative to control (P < 0.01). Hepatic total ceramide and very long chain C22:0, C24:0, C26:0 ceramide concentrations were increased during TG infusion (~72%; P < 0.05). TG infusion also enhanced hepatic very long chain GlcCer and LacCer levels (e.g., C22:0 GlcCer; P < 0.05). However, hepatic C16:0 and C18:0 ceramide levels were not modified by treatment. In support of enhanced de novo synthesis of very long chain ceramides during TG infusion, C22:0 and C24:0 dihydroceramide concentrations were elevated 70–110% (P < 0.05). Moreover, C22:0 and C24:0 ceramide were positively correlated with their dihydroceramide precursors (r ≥ 0.88, P < 0.01). Bovine liver primarily expressed CerS2 and CerS6, and TG selectively enhanced CerS2 mRNA by 60% (P < 0.05). We conclude that hyperlipidemia promotes the de novo synthesis and accumulation of very long chain ceramides in dairy cattle experiencing hepatic steatosis.