Background Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase produces reactive oxygen species (ROS) involved in oxidative stress and signal transduction. Recent studies have suggested that NADPH oxidase is associated with the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). The aim of this study was to detect the expression of NADPH oxidase subunits and 4-hydroxynonenal (4-HNE) in nasal polyp tissue and normal nasal mucosa, in order to explore the possible role played by NADPH oxidase in the pathogenesis of CRSwNP. Methods Thirteen patients with CRSwNP and 9 normal control subjects were selected to participate in this study, in which we evaluated the expression of different NADPH oxidase subunits (gp91(phox), p67(phox), p47(phox), and p22(phox)) in nasal polyp (NP) tissue and control mucosa by Western blotting and real-time polymerase chain reaction (PCR). Immunohistochemistry and immunofluorescence staining were used to detect expression of the p67(phox) subunit and 4-HNE in NP tissue and normal nasal mucosa. Results Western blot and real-time PCR results showed that p67(phox) expression was significantly increased in NP tissue when compared with its expression in control mucosa (p = 0.004). p67(phox) was expressed in the eosinophils and neutrophils found in NP tissue, but not in the macrophages. Additionally, the levels of 4-HNE expression were also significantly increased in NP tissue when compared with control mucosa (p = 0.001). Conclusion The levels of p67(phox) messenger RNA (mRNA) and protein as well as 4-HNE were both upregulated in NP tissue, suggesting that p67(phox) and oxidative stress play roles in the pathogenesis of CRSwNP.