Muscle atrophy, characterized by decreased cell number and size, is a serious concern for patients afflicted with inflammatory diseases. Mounting evidence indicates that tumor necrosis factor alpha (TNF-α) plays a critical role in muscle atrophy in a number of clinical settings. We hypothesize that reactive oxygen species (ROS) mediate TNF-α-induced muscle cell death and hypotrophy. Recently, melatonin has attracted attention because of its free-radical scavenging and antioxidant properties. The aim of the current study was to evaluate the possible protective role of melatonin in TNF-α-induced muscle cell death and hypotrophy in rat L6 myotubes. To examine this possible role, L6 myotubes were exposed to various concentrations of recombinant TNF-α for 24 hr. We found that TNF-α at a concentration of 100 ng/mL induced ROS generation and decreased cell viability. Further analysis revealed that apoptosis, but not autophagy, may be important for TNF-α-induced cell death. Melatonin significantly attenuated TNF-α-induced ROS generation and apoptosis. In addition, decreased muscle fiber diameter and increased muscle cell proteolysis by TNF-α was highly attenuated by treatment with melatonin. The effects of melatonin were mediated neither through its plasmalemmal receptors nor by modulating the nuclear factor kappa B pathway activated by TNF-α. Taken together, these results suggest that TNF-α may mediate ROS-induced muscle cell death and hypotrophy and that melatonin may be a useful tool for protecting against muscle atrophy stemming from inflammatory diseases.