When cultured in low serum containing growth medium the mouse C2C12 cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain (MHC)-positive bona fide multinucleated muscle cells. To gain an understanding into this process we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C2C12 cells and the fully differentiated myotubes by the combined methods of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), quantitative PDQuest image analysis and mass spectrometry (MS). Quantification of more than 2000 proteins from C2C12 myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C2C12 myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C2C12 myoblasts into myotubes. We identified the differentially expressed proteins by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization-quadrupole ion trap tandem mass spectrometry (LC-ESI-QIT-MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation.