Reduced insulin action is an adaptation that develops during early lactation to enhance lipolysis and promote glucose partitioning for the mammary synthesis of milk. The onset of lactation is accompanied by the accumulation of ceramides, antagonists of insulin signaling, which have been reported to be enhanced by palmitic acid (C16:0) feeding in monogastrics. To determine whether C16:0 feeding increases plasma ceramides during early lactation, 37 multiparous cows were assigned to 1 of 3 treatments: CON-CON (control diet with no supplemental fat; 1–67 DIM); CON-PA (control diet fed from 1 to 24 DIM, and a C16:0-supplemented diet fed from 25 to 67 DIM); and PA-PA (C16:0 supplemented diet; 1–67 DIM). Diets were formulated to contain a minimum of 17% CP, 29% NDF, and 24% starch. The C16:0 supplement (85% C16:0), added at 1.5% of diet DM, replaced soyhulls in the CON diets. Blood was collected at 11, 32, and 60 DIM. Plasma ceramide and monohexosylceramide (GlcCer) concentrations were determined using mass spectrometry. Data were analyzed as repeated measures using a mixed model, and Pearson correlations were analyzed. Circulating ceramides increased with the progression of lactation (P < 0.01), albeit the magnitude was greater with PA feeding (day by treatment interaction; P < 0.05). PA increased total ceramide levels by 39% (P < 0.01). The addition of C16:0 in CON-PA increased total ceramides by 60% at d 32, relative to d 11 (P < 0.01). Relative to CON-CON, PA-PA increased C24:0-ceramide levels by 26, 48, and 58% at d 11, 32, and 60, respectively (P < 0.05). The ratio of C24:0- to C16:0-ceramide decreased over time and was elevated in PA-PA (P < 0.05). The majority of GlcCer increased over time, and were elevated in PA-PA, relative to CON-CON (P < 0.01). For example, total GlcCer increased 25% from d 11 to 60, and was 25% higher in PA-PA, relative to CON-CON (P < 0.01). Plasma total ceramide and C24:0-ceramide levels were positively correlated with milk yield and energy-corrected milk (r = 0.27 to 0.52; P < 0.01). The ability of palmitic acid-induced ceramide to suppress insulin action during early lactation requires further investigation.