Genetic or biochemical abnormalities in mitochondria are closely associated with apoptosis, aging, cancer, and other chronic degenerative diseases. Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) depletion dispatches retrograde signals to the nucleus to compensate by altering the expression of various genes. In this study, a proteomic approach was used to gain insight into the nuclear gene targets of mitochondrial stress signaling and the pathophysiological mechanisms associated with mitochondrial dysfunction. We have used 2-DE to characterize the nuclear gene responses resulting from mtDNA depletion in L6 GLUT4myc myocytes. Our results showed that 77 polypeptides were differentially expressed in mtDNA-depleted cells; 33 polypeptides were down-regulated and 44 polypeptides were up-regulated. Of these differentially expressed polypeptides, 40 were identified as 36 different proteins by MALDI-TOF MS. These proteins are related to various cellular responses, such as apoptosis, cellular metabolism, signaling and cytoskeleton functions. It is suggested that the insulin resistance developed in mtDNA-depleted myocytes may be associated with disorganization of cytoskeleton assembly, and that cellular mtDNA depletion might promote the ability to evade apoptosis or other death effectors.