Saturated fatty acids can antagonize insulin sensitivity in rodents by increasing ceramide (Cer) levels in liver and plasma. Cer can be generated via de novo synthesis, sphingomyelin (SM) hydrolysis, or sphingolipid recycling. In turn, Cer can be glycosylated to form monohexosylceramide (GlcCer) and lactosylceramide (LacCer). Analyzing the ratio between sphingolipids, within identical acyl chain length and saturation, can be a means to understand Cer metabolism. Our objective was to evaluate whether an increase in plasma NEFA can modify the sphingolipid ratio 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. Blood was collected at routine intervals. LC/MS was used to profile sphingolipids in plasma. Log-transformed data were analyzed using a mixed model with repeated measures. 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. Ratio of C16:0-SM to C16:0-Cer (C16:0 SM:Cer) decreased 9% by 3 h of TG infusion, relative to control (P < 0.05). C22:0, C22:1, and C24:0 SM:Cer decreased 27, 52, and 17% by 16 h of TG infusion, relative to control (P < 0.01). TG infusion did not modify C16:0-dihydro SM:Cer; however, TG-infusion lowered the ratio of C18:0-SM to C18:0-dihydro-SM (P < 0.01). C16:0 and C18:0 Cer:GlcCer decreased 24 and 13% by 16 h of TG infusion, relative to control (P < 0.01). In contrast, C24:0 Cer:GlcCer and C26:0 Cer:GlcCer increased with TG infusion, relative to control (P < 0.05). C18:0, C22:0, and C24:1 GlcCer:LacCer increased in TG-infused cows, relative to control (P < 0.01). C16:0 Cer:GlcCer and C24:0 SM:Cer were negatively correlated with NEFA, and C24:0 Cer:GlcCer was positively correlated with NEFA (P < 0.01). Increasing plasma NEFA lowers the ratio of SM to Cer in dairy cows, consistent with a shift in metabolism toward Cer accumulation.