Insulin resistance in rodents is characterized by inflammation-mediated activation of sphingomyelinase and the transformation of sphingomyelin (SM) into ceramide (Cer), an effect provoked by saturated fatty acids. Our objective was to determine whether an acute increase in circulating NEFA lowers plasma SM levels in cows. Six nonpregnant, 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. LC/MS was used to profile SM in plasma. Data were analyzed using a mixed model with repeated measures (fixed effects of treatment and time). Nonparametric correlations were analyzed. TG infusion increased plasma NEFA by 454% at 3 h relative to control (P < 0.01) with no further increase at 16 h. Total plasma Cer concentrations increased in TG-infused cows, relative to control (P < 0.01). Before infusion, C16:0- and C18:1-SM represented 30 and 21%, respectively, of plasma SM. Plasma C16:0-SM levels increased 10% by 16 h of TG infusion, relative to control (P < 0.05). Plasma C20:1-SM concentrations were lower in TG-infused cows, relative to control (P < 0.05). Plasma C16:1-, C18:0-, C18:1-, C22:0-, C24:0-, and C24:1-SM, as well as total plasma SM were not modified with TG infusion. Before infusion, C20:0-dihydro-SM (DHSM) represented 89% of total plasma DHSM. Plasma C20:0- and C18:0-DHSM increased 79 and 14%, respectively, by 16 h of TG infusion, relative to control (P < 0.01). Plasma C18:1- and C20:1-SM were negatively correlated with NEFA (e.g., C20:1-DHSM, r = −0.47; P < 0.01). In contrast, plasma C18:0- and C20:0-DHSM, as well as total DHSM were positively correlated with NEFA (e.g., C20:0-DHSM, r = 0.77; P < 0.01). An acute increase in plasma NEFA does not appear to promote sphingomyelin hydrolysis in dairy cows thus suggesting that plasma Cer accumulations occurred due to enhanced de novo Cer synthesis.