--- - |2- Hepatic insulin resistance induced by high‐fat diet resulted from an increase in synthesis rate of ceramide and diacylglycerol (DAG). Myriocin treatment improved insulin sensitivity, which was accompanied by a decrease in the synthesis rate and content of ceramide and DAG. Liver, as one of the most important organs involved in lipids and glucose metabolism, is perceived as a key tissue for pharmacotherapy of insulin resistance (IRes) and type 2 diabetes. Ceramides (Cer) are biologically active lipids, which accumulation is associated with the induction of muscle IRes. We sought to determine the role of intrahepatic bioactive lipids production on insulin action in liver of insulin‐resistant rats and after myriocin administration. The experiments were conducted on male Wistar rats divided into three groups: Control, fed high‐fat diet (HFD), and fed HFD and treated with myriocin (HFD/Myr). Before sacrifice, the animals were infused with a [U‐13C]palmitate to calculate lipid synthesis rate by means of tracer incorporation technique in particular lipid groups. Liver Cer, diacylglycerols (DAG), acyl‐carnitine concentration, and isotopic enrichment were analyzed by LC/MS/MS. Proteins involved in lipid metabolism and insulin pathway were analyzed by western blot analysis. An OGTT and ITT was also performed. HFD‐induced IRes and increased both the synthesis rate and the content of DAG and Cer, which was accompanied by inhibition of an insulin pathway. Interestingly, myriocin treatment reduced synthesis rate not only of Cer but also DAG and improved insulin sensitivity. We conclude that the insulin‐sensitizing action of myriocin in the liver is a result of the lack of inhibitory effect of lipids on the insulin pathway, due to the reduction of their synthesis rate. This is the first study showing how the synthesis rate of individual lipid groups in liver changes after myriocin administration. - 'Journal of Cellular Physiology, Volume 234, Issue 2, Page 1851-1861, February 2019. '