Respiratory muscle weakness resulting from both diaphragmatic contractile dysfunction and atrophy has been hypothesized to contribute to the weaning difficulties associated with prolonged mechanical ventilation (MV). While it is clear that oxidative injury contributes to MV-induced diaphragmatic weakness, the source(s) of oxidants in the diaphragm during MV remain unknown. These experiments tested the hypothesis that xanthine oxidase (XO) contributes to MV-induced oxidant production in the rat diaphragm and that oxypurinol, a XO inhibitor, would attenuate MV-induced diaphragmatic oxidative stress, contractile dysfunction, and atrophy. Adult female Sprague-Dawley rats were randomly assigned to one of six experimental groups: 1) control, 2) control with oxypurinol, 3) 12 h of MV, 4) 12 h of MV with oxypurinol, 5) 18 h of MV, or 6) 18 h of MV with oxypurinol. XO activity was significantly elevated in the diaphragm after MV, and oxypurinol administration inhibited this activity and provided protection against MV-induced oxidative stress and contractile dysfunction. Specifically, oxypurinol treatment partially attenuated both protein oxidation and lipid peroxidation in the diaphragm during MV. Further, XO inhibition retarded MV-induced diaphragmatic contractile dysfunction at stimulation frequencies >60 Hz. Collectively, these results suggest that oxidant production by XO contributes to MV-induced oxidative injury and contractile dysfunction in the diaphragm. Nonetheless, the failure of XO inhibition to completely prevent MV-induced diaphragmatic oxidative damage suggests that other sources of oxidant production are active in the diaphragm during prolonged MV.