The development of insulin resistance represents a homeorhetic adaptation to support lactation in dairy cattle; however, impaired insulin action can be a detriment to metabolic health during the peripartum. To delineate the mechanisms of insulin resistance, we have focused on the sphingolipid ceramide which is an antagonist of insulin action in diabetic monogastrics. A lipidomics workflow that includes the application of targeted and untargeted mass spectrometry has been employed to investigate the relationship between ceramide metabolism and insulin sensitivity in dairy cattle. Studies have established that the peripartal progression of hyperlipidemia, hepatic lipid accumulation, and insulin resistance is accompanied by hepatic and circulating ceramide accumulation. Although these changes develop in all cows, enhanced adiposity predisposes cows to heightened ceramide accrual which may explain their observed impairment in lipolytic responsiveness to glucose. Hepatic de novo ceramide synthesis and export can respond to increased fatty acid delivery and altered mitochondrial fatty acid processing. Specifically, the induction of hyperlipidemia by intravenous triacylglycerol infusion or by nutrient-restriction can increase de novo ceramide synthesis in dairy cattle. However, ceramide pools may also rise in response to inflammation-provoked sphingomyelin hydrolysis. Importantly, the ability of ceramide to antagonize insulin action is influenced by the acyl moiety and glycosylation status. Our series of lipidomic studies have revealed circulating lignoceroyl sphingosine (C24:0-ceramide) as an insulin resistance biomarker. Feeding regimens utilized on-farm can influence C24:0-ceramide supply. For example, palmitic acid feeding increases C24:0-ceramide supply in lactating dairy cows, relative to stearic acid or no fat supplementation. Also, micronutrients nicotinic acid and choline can influence ceramide levels. On-going research is investigating novel pharmacological and nutritional approaches to modify ceramide synthesis and insulin sensitivity as a means of improving dairy cow health and performance.