The progression of insulin resistance in dairy cows represents a maternal adaptation to support milk production during heightened energy demand; however, excessive adipose tissue lipolysis can develop. In diabetic monogastrics, the mechanisms that mediate insulin resistance involve the sphingolipid ceramide (cer). We tested the hypothesis that ceramide accumulates in dairy cows experiencing lipolysis and insulin resistance. Nine non-pregnant, nonlactating dairy cows were utilized in a replicated 3 × 3 Latin square design. Cows were ad libitum fed, nutrient-restricted (NR), or NR with nicotinic acid (NA; 5 mg NA/h per kg BW i.v.) for 34 h. Ad libitum fed cows were provided a mixed ration to meet or exceed requirements. Intake for NR or NR + NA cows was limited to vitamins and minerals. At the end of treatment, a liver biopsy and insulin challenge were performed. Before and after treatment, all cows were provided ad libitum access to feed. Serum was analyzed using colorimetry and mass spectrometry. Data were analyzed using a mixed model with repeated measures. As previously established, NR progressively increased serum fatty acid levels (69 vs. 725 µmol/L at h 32; P < 0.01) and decreased glucose clearance rate (CR_{40 min}; 1.76 vs. 1.36%/min; P < 0.01). Feed removal increased liver lipid content by 35% (P < 0.01). Although lipolysis was suppressed 55% in cows infused NA (P < 0.01), CR_{40 min} was not improved relative to NR cows. Notably, NR increased serum 16:0- and 24:0-cer 75 and 78% by h 32 (P < 0.01). Similar responses were observed for 18:0-, 20:0-, and 22:0-cer (P < 0.05). NA was unable to prevent the rise in ceramide. NR with or without NA increased hepatic ceramide (56 to 116%; P < 0.01) relative to feeding. Re-feeding lowered ceramide levels (P < 0.05). We detected an inverse relationship between very long chain ceramide levels and insulin sensitivity (e.g., serum and hepatic 24:0-cer vs CR_{40 min}, r = −0.51, P < 0.01), a response not observed for 16:0-, 18:0-, 20:0-cer, or fatty acids. Our data support a role for very long chain ceramides as antagonists of insulin sensitivity in dairy cows experiencing energy deficit.