Lipid oversupply is closely linked to the development of insulin resistance. We have identified two members of the lipid-activated Protein Kinase C (PKC) family, PKCδ and PKCε, which play a part not only in the modulation of insulin action but also in the regulation of hepatic lipid accumulation. After one week of high fat feeding, both PKCδ knockout (PKCδ-KO) and PKCε knockout (PKCε-KO) mice displayed improvement in glucose tolerance and protection against insulin resistance compared to wild type (WT) mice. Surprisingly, despite this similarity, fat-fed PKCδ-KO mice accumulated less triglyceride in the liver while PKCε-KO mice accumulated more. We investigated the molecular mechanisms by which PKCδ and PKCε exert similar effects on insulin action yet opposite effects on hepatic lipid metabolism by conducting quantitative proteomics profiling. Using an in vivo adaptation of the SILAC-labelling and LC-MS/MS, 1% of the approximately 3000 proteins detected for each PKC isoform were either upregulated or downregulated in liver from fat-fed PKC-deficient mice compared to WT mice. These included enzymes of lipid, cholesterol and amino acid metabolism, proteins linked to redox stress and also cytoskeletal and nuclear proteins. Interestingly, 10 proteins were regulated by both PKC isoforms, either in a similar or converse fashion. A possible interpretation is that proteins displaying opposite regulation, such as the long chain acyl-CoA synthase 4 binding partner, Htatip2, are associated with the altered lipid metabolism mediated by deletion of the kinases. Meanwhile, those exhibiting similar changes, such as three isoforms of flavin-containing monooxygenase, may be involved in protection against lipid-induced insulin resistance. By further examination of these candidates from our hepatic proteomic studies, we will gain novel insights into PKC-dependent regulation of insulin action and lipid metabolism.