Dairy cows transitioning from gestation to lactation develop insulin resistance to partition nutrients to the mammary gland to support lactation. Reduced insulin action promotes lipolysis, leading to increased free fatty acids (FFA) in circulation. Free fatty acids are used as a substrate to de novo synthesize the sphingolipid ceramide, which is important because ceramides cause insulin resistance in nonruminants. We have previously observed that ceramides are positively associated with insulin resistance and milk yield in lactating ruminants. It has not been determined if ceramides directly mediate this response. In the first experiment herein, we tested the hypothesis that the mechanism recombinant bovine somatotropin (rBST) uses to increase milk production involves ceramide. We observed that rBST administration increased very-long-chain (VLC) circulating ceramide concentrations in cows in association with increased FFA and milk yield, during a time when insulin-stimulated glucose disposal tended to be lower. We also characterized the plasma lipidome and determined that plasma di- and triacylglycerols levels, phosphatidylcholines, and sphingomyelins were reduced. These data support a role for ceramide in somatotropin-induced milk production. In the second experiment, we investigated whether ceramide synthesis is suppressed by the serine palmitoyltransferase inhibitor myriocin and increased by nutrient restriction in ewes. Nonlactating ewes received an intravenous bolus of myriocin (0, 0.1, 0.3, or 1.0 mg/kg/body weight [BW]; CON, LOW, MOD, or HIGH) every 48 h for 17 d. We observed an appetite-suppressing effect of myriocin in HIGH. Myriocin linearly decreased plasma VLC Cer and dihydroceramide (DHCer) by d 13. Nutrient restriction increased plasma C16:0-Cer, an effect suppressed by MOD and HIGH. Myriocin linearly decreased most Cer and DHCer species in liver and omental and mesenteric adipose, VLC Cer and DHCer in pancreas, and C18:0-Cer in skeletal muscle and subcutaneous adipose tissue. We conclude intravenous delivery of 0.3 mg of myriocin/kg of BW/48 h for 17 d decreases circulating and tissue ceramide concentrations without modifying energy intake in ruminants. Future research should investigate whether nutritional or pharmacological control of ceramide synthesis influences the ability of rBST to promote milk synthesis, and should test the hypothesis that elevated ceramide concentrations are required for milk production.