Prolonged disuse of skeletal muscle causes atrophy, which is a universal phenomenon induced by various factors such as cast immobilization and space flight. However, the identity of proteins produced in atrophying skeletal muscle cells is poorly understood, especially during a period of cast immobilization. In the present study, we used 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry to investigate protein expression in rat gastrocnemius subjected to cast immobilization for 7 and 21 days. Gastrocnemius muscle mass was lost continuously throughout the 21 days of cast immobilization. Proteomic analysis of silver-stained gels of whole-protein extracts from rat gastrocnemius muscle strips detected 48 proteins. Of these proteins, the expression of 6 proteins changed during cast immobilization; these proteins are involved in metabolic, contraction, and chaperone activities. These results suggest that cast immobilization-induced skeletal muscle atrophy is related to changes in the defense and contractile apparatus proteins in rat gastrocnemius muscle.