Lipoprotein ceramide can antagonize insulin signaling. We have previously demonstrated increased circulating ceramide in dairy cows transitioning from gestation to lactation; however, the origin of ceramide required validation. Therefore, our objective was to characterize the ceramide composition of bovine lipoproteins. Basal blood samples were collected from 4 non-pregnant, nonlactating Holstein dairy cows ad libitum fed a diet containing corn silage and grass hay. To fractionate triacylglycerol (TAG)-rich, low density, and large and small high density lipoproteins (VLDL, LDL, and buoyant and dense HDL, respectively) from serum, we employed fast protein liquid chromatography using a size exclusion column (10 × 300 mm; 5 to 650 kDa). Thirty 0.5 mL fractions were continuously collected and analyzed for TAG, phospholipid, total cholesterol, and protein levels using colorimetry. In turn, fractions corresponding to VLDL, LDL, and buoyant and dense HDL were pooled (1.5 mL). Following extraction, ceramide levels within whole serum and pooled fractions were quantified using mass spectrometry. Data were analyzed using a mixed model with repeated measures. Results (presented relative to all other pooled fractions) demonstrate that VLDL primarily contained TAG (2.2 mg/dL; 50.9% of total components; P < 0.01). Low density lipoproteins exhibited the greatest concentrations of cholesterol and phospholipid (15.6 and 15.5 mg/dL, respectively; P < 0.01). Buoyant HDL contained elevated levels of cholesterol, phospholipid, and protein (6.8, 6.7, and 8.2 mg/dL, respectively; P < 0.01). In contrast, dense HDL primarily contained protein (5.4 mg/dL; P < 0.01). Our results confirm that LDL are enriched with ceramide (P < 0.01); although, ceramide was compartmentalized to a lesser extent within both HDL subclasses and VLDL. Comparable to whole serum, C16:0-ceramide was the predominant ceramide quantified. Interestingly, the proportion of C24:0-ceramide to total ceramide was elevated in VLDL (P < 0.01). We conclude that bovine LDL are enriched with ceramide, and lipoprotein ceramide profiles mimic levels quantified in whole serum. Future research must determine the biological importance of lipoprotein ceramides.