Prevention of oxidative stress via antioxidants attenuates diaphragm myofiber atrophy associated with mechanical ventilation (MV). However, the specific redox sensitive mechanisms responsible for this remain unknown. We tested the hypothesis that regulation of skeletal muscle proteolytic activity is a critical site of redox action during MV. Sprague-Dawley rats were assigned to five experimental groups: 1) control (Con): 2) 6-hours of MV (6hr MV); 3) 6-hours of MV with infusion of the antioxidant, Trolox (6hr MVT), 4) 18-hours of MV (18hr MV); and 5) 18-hours MV with Trolox (18hr MVT). Trolox did not attenuate MV induced increases in diaphragmatic levels of ubiquitin-protein conjugation, poly-ubiquitin mRNA, and gene expression of proteasomal subunits (20S proteasome -subunit 7, 14kDa-E2, and proteasome-activating complex PA28). However, Trolox reduced both chymotrypsin-like and PGPH-like 20-S proteasome activities in the diaphragm following 18hr of MV. In addition, Trolox rescued diaphragm myofilament protein concentration (µg/mg muscle) and the percentage of easily releasable myofilament protein independent of alterations in ribosomal capacity for protein synthesis. In summary, these data are consistent with the notion that the protective effect of antioxidants on the diaphragm during MV is due, at least in part, to decreasing myofilament protein substrate availability to the proteasome.