In this study we used a mass spectrometry based targeted lipid omics approach to study the effect of WD and myriocin on hepatic Q-VD-OPh ceramide metabolism, which enabled us to assess changes indifferent ceramide species and total ceramides. Distinct ceramide species are synthesized in mammals by a family of six ceramide synthases. C16:0 and C24:0 ceramides are generated by CerS5/6 and CerS2, respectively. The acyl chain length defines different functions of distinct ceramide species. For example, overexpression of CerS5 promotes apoptosis while overexpression of CerS2 protects from apoptosis. In addition, CerS2 deficiency in mice results in severe hepatopathy with an increased rate of hepatic apoptosis from~30days of age and progressive hepatomegaly and hepatocellular carcinoma from~10 months of age. These data suggest that C24:0 ceramide plays acritical role in liver homeostasis. On the other hand, CerS6 deficiency-induced suppression of C16:0 ceramide in mice protects against insulin resistance. In contrast, the increase of C16:0 ceramide by overexpression of CerS6 in isolated primary hepatocytes results in triglyceride accumulation, inhibition of insulin signaling and altered mitochondrial function; all characteristics observed in NAFLD. Finally, it has been demonstrated that liver-specific ablation of CerS2 in mice causes hepatic insulin resistance due to reduced phosphorylation of the insulin receptor and its inability to translocate into detergent resistant membranes. This was TCID associated with altered plasma membrane composition due to reduced C24:0 ceramide levels. Collectively, these findings highlight causal roles for bothC16:0 and C24:0 ceramides in insulin resistance and hepatic homeostasis. Importantly, we found that a WD leads to increased levels of C16:0 but reduced levels of C24:0 in the liver. It is expected that the high palmitate content of a WD and insulin resistance-induced lipogenesis lead to increased production of C16:0 ceramide. However, it is unclear why C24:0 ceramide was reduced on a WD.One possible reason for these bidirectional changes in hepatic content maybe related to sequestration of asphingoid precursor with excessive palmitoyl-CoA that results in limited precursor availability for C24:0 ceramide synthesis.