Mechanical ventilation (MV) is a clinical tool that provides respiratory support to patients unable to maintain adequate alveolar ventilation on their own. Although MV is often a life-saving intervention in critically ill patients, an undesired side-effect of prolonged MV is the rapid occurrence of diaphragmatic atrophy due to accelerated proteolysis and depressed protein synthesis. Investigations into the mechanism(s) responsible for MV-induced diaphragmatic atrophy reveal that activation of the calcium-activated protease, calpain, plays a key role in accelerating proteolysis in diaphragm muscle fibers. Moreover, active calpain has been reported to block signaling events that promote protein synthesis (i.e., inhibition of mammalian target of rapamycin (mTOR) activation). While this finding suggests that active calpain can depress muscle protein synthesis, this postulate has not been experimentally verified. Therefore, we tested the hypothesis that active calpain plays a key role in the MV-induced depression of both anabolic signaling events and protein synthesis in the diaphragm muscle. MV-induced activation of calpain in diaphragm muscle fibers was prevented by transgene overexpression of calpastatin, an endogenous inhibitor of calpain. Our findings indicate that overexpression of calpastatin averts MV-induced activation of calpain in diaphragm fibers and rescues the MV-induced depression of protein synthesis in the diaphragm muscle. Surprisingly, deterrence of calpain activation did not impede the MV-induced inhibition of key anabolic signaling events including mTOR activation. However, blockade of calpain activation prevented the calpain-induced cleavage of glutaminyl-tRNA synthetase in diaphragm fibers; this finding is potentially important because aminoacyl-tRNA synthetases play a central role in protein synthesis. Regardless of the mechanism(s) responsible for calpain's depression of protein synthesis, these results provide the first evidence that active calpain plays an important role in promoting the MV-induced depression of protein synthesis within diaphragm fibers.