Ceramide mediates the development of insulin resistance, and the hepatic synthesis of ceramide is promoted by saturated fatty acids (FA). Feeding palmitic acid to lactating cows increases plasma ceramides, relative to a non-added fat control. Our objective was to evaluate the relationship between saturated FA chain length and circulating ceramide concentrations. Eleven cannulated Holstein cows (150 ± 52 DIM) were administered continuous abomasal infusions (280 g/d) of palmitic acid (PA; 85% C16:0), stearic acid (SA; 98% C18:0), or medium-chain triglycerides (MCT; C8:0/C10:0) for 7 d in a replicated Latin square design. Blood was collected on d 5–7 and liver biopsied on d 7 of each period. Plasma ceramide, monohexosylceramide (GlcCer), and lactosylceramide (LacCer) levels were determined using mass spectrometry. Plasma free FA levels and hepatic gene expression were evaluated. Data were analyzed under a mixed model. Orthogonal contrasts compared PA vs. SA and PA vs. MCT, and correlation analysis performed. As established, PA infusion increased milk fat yield and fat-corrected milk (FCM) feed efficiency relative to SA (6.5 and 9.3%, respectively; P < 0.05). Relative to SA, PA increased plasma free FA by 21% (P < 0.05). Infusing PA increased plasma total ceramide and GlcCer levels relative to SA and MCT (~21%, P < 0.05). For example, PA increased C24:0-ceramide by 28 and 36% relative to SA and MCT, respectively (P < 0.01). Most pronounced, PA increased C26:1-ceramide by 46%, relative to SA (P < 0.01). Plasma ceramide, GlcCer, and LacCer levels were positively associated with plasma free FA, and yields of milk and FCM (r = 0.3–0.65; P < 0.05). Although not influenced by infusate, hepatic carnitine acyltransferase I and apolipoprotein-B100 mRNA expression were inversely associated with plasma ceramide and GlcCer levels (r = 0.32–0.72; P < 0.05). We conclude that saturated FA chain length can influence ceramide levels in relation to hepatic gene transcription. The preferential ability of palmitic acid to induce ceramide synthesis is likely due to the selective requirement of serine palmitoyltransferase for palmitoyl-CoA.