Erectile dysfunction (ED) is a frequent occurrence in male patients with obstructive sleep apnea syndrome (OSAS). Long-term intermittent hypoxia (LTIH), one of the hallmarks of OSAS, could mediate ED. The objective of this study was to test the hypothesis that increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity contributes to ED in rat responses to LTIH. Healthy male Sprague-Dawley rats were randomly distributed into 4 groups: a LTIH group, an apocynin (a selective NADPH oxidase inhibitor)-treated LTIH group, a sham LTIH group, and an apocynin-treated sham group. Erectile function was examined by measuring the mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) on electrical stimulation of the cavernous nerve. Real-time quantitative polymerase chain reaction and Western blot were used to examine mRNA and protein expression of NADPH oxidase subunit in corpus cavernosa (CC). The level of malondialdehyde and superoxide dismutase were detected by colorimetry. Nitric oxide synthase (NOS) isoforms in CC were also investigated. LTIH markedly attenuated the erectile responses (ICP/MAP), and these were partially prevented by apocynin treatment. Promoted oxidative stress associated NADPH oxidase subunit activation was found in CC from LTIH rats. Decreased expression and activity of constitutive NOS (cNOS), including endothelial NOS and neuronal NOS, associated with enhanced inducible NOS (iNOS) expression and activity were observed in LTIH rats. Apocynin prevented the decrease in cNOS activity and inhibited iNOS expression and activity in LTIH rats. These results indicate that NADPH oxidase activation plays an important role in the pathogenesis of LTIH-mediated ED.