Control of human papillomavirus (HPV) infection involves the activation of Toll-like receptors (TLRs), key components of the mucosal antiviral response. Available studies on TLR expression in HPV-positive cervical cells are limited and reported conflicting results. This study quantified TLR 2, 3, 4, 7 and 9 transcripts in low-risk (LR) and high-risk (HR) HPV-positive and HPV-negative cervical samples from 154 women attending a gynaecological clinic. Expression levels of TLR 2, 3, 4 and 7 did not differ among samples, whereas TLR9 levels were quite significantly higher in LR and marginally significant in HR HPV-positive samples, with respect to the HPV-negative samples. Interestingly, in a subgroup of women with documented previous HPV-infection, TLR9 levels were extremely higher in patients persistently positive to the same HPV genotype for more than one year, with respect to women who cleared HPV infection and to those re-infected with a different genotype. These findings implicate TLR9 in the response to LR and HR HPVs, including HPV 16 known to interfere with TLR9 transcription in cell lines. Elevated TLR9 levels without HPV clearance in persistently infected women could drive inflammation thereby contributing to cervical cancer risk.