Abstract: OBJECTIVE To explore the expression of Toll-like receptor 4 and inducible nitric oxide synthase(TLR4/iNOS)pathway molecules in diseased tissues and cell lines of cervical cancer patients with high-risk human papillomavirus (HR-HPV) infection and analyze the related mechanisms in development of cervical cancer. METHODS Totally 40 cervical cancer patients who were confirmed with HR-HPV infection from Jan 2014 to Aug 2015 were enrolled in the study and assigned as the cervical cancer group, the cervical cancer tissues specimens were collected, and normal cervical tissues specimens were obtained from 40 patients with gynecological benign lesions who underwent hysterectomy. The cervical cancer cell lines CaSki positive for HPV 16 (4 strains), Hela positive for HPV 18 (4 strains), and C33a negative for HPV (4 strains) were purchased. The cervical cancer cell lines were cultured, and the TLRs and iNOS contents were detected by using SP immunohistochemistry. RESULTS The mRNA contents of TLR4 and iNOS were respectively (152.58±30.74)ratio and (283.05±11.13)ratio in the cervical cancer tissues of the patients with HR-HPV infection, significantly higher than (103.75±10.12)ratio and (98.50±10.84)ratio in the normal cervical tissues (P<0.05). There was no significant difference in the mRNA content of TLR3, TLR7, TLR8, or TLR9 between the cervical cancer tissues and the normal cervical tissues. The mRNA contents of iTLR4, iNOS, and NO were respectively (174.67±33.67)ratio, (312.94±24.01)ratio, and (63.94±8.53)μmol/L in the CaSki cell line and were respectively (326.48±38.55)ratio, (325.06±34.80)ratio, and (70.73±8.32)μmol/L in the Hela cell line, higher than (98.59±21.46)ratio, (99.83±23.55)ratio, and (21.56±3.51)μmol/L in the C33a cell line. CONCLUSION The expression of TLR4/iNOS pathway molecules shows significant up-regulation in both the diseased tissues and cell lines of the cervical cancer patients with HR-HPV infection, it influences the generation of NO, which may involve in the pathogenic process of cervical cancer of HR-HPV.