An increase in plasma NEFA can impair insulin sensitivity in dairy cows by unknown mechanisms. In monogastrics, excess saturated fatty acids can upregulate the hepatic synthesis and secretion of ceramide, a sphingolipid that inhibits insulin action in muscle and adipose tissue. Our objective was to determine whether an increase in plasma NEFA can augment ceramide 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 and liver was biopsied at 16 h. LC/MS was used to profile 25 ceramides (Cer), monohexosylceramides (GlcCer), and lactosylceramides (LacCer) 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. Liver TG were elevated 321% in TG vs control by end of TG infusion (P < 0.01). Before infusion, C24:0-Cer, C24:0-GlcCer, and C16:0-LacCer represented 52, 47, and 83%, respectively, of plasma Cer, GlcCer and LacCer. Plasma total Cer increased 149 and 235% by 3 and 16 h of TG infusion, relative to control (P < 0.01). Plasma C20:0-, C22:0-, C24:0-, and C26:0-Cer levels increased during TG infusion (P < 0.01). Although TG infusion did not modify plasma C16:0-Cer level at 16 h, C16:0-Cer abundance (% of total) was lower at 16 h (P < 0.01). TG infusion increased plasma C16:0-, C20:0-, C22:0-, and C24:0-GlcCer levels (P < 0.01). Plasma LacCer levels were not modified by TG infusion. Cer levels were positively correlated with NEFA (e.g., C24:0-Cer, r = 0.80; P < 0.01). Our data indicate that plasma Cer accumulation occurs in dairy cows concomitantly with increases in plasma NEFA and liver TG. Research is needed to determine whether NEFA-induced insulin resistance is mediated by Cer.