Introduction Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common and frequently occurring disease of the upper respiratory tract. The nasal instillation of the Gram‐negative (G−) bacterial product lipopolysaccharide (LPS) can induce not only acute sinusitis but also the development of CRSwNP in animal models. Nevertheless, the expression and distribution of LPS in patients with CRSwNP have not been investigated. And the study was to investigate the expression of LPS and its relationship with glucocorticoid receptors (GRs) in CRSwNP. Methods Multiple methods, including bacterial culture and immunohistochemistry, were used to detect and analyze nasal bacteria, plasma LPS content, and the levels of LPS and GR‐α/β, cluster of differentiation 68 (CD68), and myeloperoxidase (MPO) expression, as well as their relationship in CRSwNP. Results The number of G− bacteria and Escherichia coli (E. coli) was not significantly different between CRSwNP subjects and the controls. However, the positive rate of LPS was much higher than that of E. coli in CRSwNP subjects and was significantly higher in noneosinophilic CRSwNP subjects than in eosinophilic CRSwNP subjects. Moreover, the LPS levels were positively correlated with GR‐β but not GR‐α expression in CRSwNP. Immunofluorescence assays showed that LPS was mainly detected in CD68+ macrophages and MPO+ neutrophils, in addition to histiocytes, in CRSwNP. Conclusions Persistent LPS in CRSwNP can lead to unresolved mucosal inflammation, eventually leading to tissue remodeling and the development of CRSwNP. Our findings suggest that increased LPS content and possible resistance to glucocorticoids may be one of the important pathogenic mechanisms of G− bacteria in CRSwNP.