The purpose of this study was to determine whether Toll-like receptor 5 (TLR5) expression was associated with disease progression in cervical neoplasia. TLR5 expression was evaluated by immunohistochemistry (IHC) in 55 formalin-fixed paraffin-embedded cervical tissues; 10 normal cervical specimens, 9 low-grade cervical intraepithelial neoplasias (CINs), 12 high-grade CINs, and 24 invasive squamous cell carcinomas (ISCCs). TLR5 expression was also evaluated at the RNA level, in fresh, frozen cervical carcinoma tissues by real-time quantitative RT-PCR. TLR5 expression, which was mainly observed as cytoplasmic staining, gradually increased in accordance with the histopathologic grade in the following order: low-grade CIN less than high-grade CIN less than ISCC (P < 0.001). Immunohistochemical staining showed that TLR5 expression was undetectable (80%) or weak (20%) in normal cervical squamous epithelial tissues. However, moderate expression was detected in 33.3% of low-grade CIN (3/9), 41.7% of high-grade CIN (5/12), and 45.8% of ISCC (11/24). Strong expression was detected in as much as 33.3% of high-grade CIN (4/12) and 50% of ISCC (12/24). Contrary to IHC results, real-time quantitative RT-PCR revealed that TLR5 expression in tumors was not statistically different compared to normal cervical tissues (P = 0.1452). The IHC result suggests that TLR5 may play a significant role in tumor progression of cervical neoplasia and may represent a useful marker for malignant transformation of cervical squamous cells.