The Cbl family of proteins are multidomain proteins that have a dual function, as a protein adaptors for tyrosine kinase receptors and as E3-ubiquitin conjugating enzymes. As such, they perform a variety of functions in different tissues and in response to various stimuli. In adipocytes and skeletal muscle c-Cbl is rapidly phosphorylated in response to insulin both in vitro and in vivo (Gupte,2006). In adipocytes, c-Cbl is found associated with CAP (Cbl Associated protein) which helps recruit c-Cbl to the lipid raft microdomains of the plasma membrane. c-Cbl null animals were reported to be more insulin sensitive and be resistant to the deleterious effects of a high fat diet (Molero,2004). The aim of this study was to understand the function of Cbl and CAP in muscle cells and their contribution to cell metabolism. Cbl or CAP were constitutively knocked down in C2C12 cells using lentiviral transduction of specific shRNAs. Western blot analysis were carried out to determine activation of insulin and nutrient signalling pathways in control and shRNA-expressing cells. Cellular oxygen consumption was determined with a Seahorse XF bioanalyser and ATP levels and activity of OXPHOS were determined in vitro using spectrophotometric and bioluminescent methods. Native blue gel electrophoresis (1D and 2D) were used to determine the assembly of OXPHOS mitochondrial complexes. We found that Cbl KD cells displayed normal activation of phosphatidylinositoI 3-kinase /AKT and ERK pathways in response to insulin. However, ATP levels were reduced compared to control cells. Consistent with this, AMP-regulated kinase phosphorylation levels were elevated in Cbl KD cells. Maximal mitochondrial oxygen consumption was reduced in Cbl KD cells, together with reduced activation of long chain fatty acyl-COA formation. In vitro, the activity of mitochondrial OXPHOS complexes I, IV and V were reduced compared to that of non-targeting shRNA-expressing cells. Consistent with these findings, we found that the assembly of these proteins into supercomplexes SCI+III+IV and SC III+IV was reduced in Cbl KD cells compared to controls. Overall, our data demonstrates that c-Cbl depletion in muscle cells results in mitochondrial impairment and suggest that this may be due in part to a reduced capacity for OXPHOS complex assembly into supercomplexes.