Interferons (IFNs) are expressed by many cell types and play a pivotal role in the generation of immune responses against viral infections. IFN-κ, a novel type I IFN, displays a tight tropism for keratinocytes and specific lymphoid populations and exhibits functional similarities with other type I IFNs. The human papillomavirus (HPV), the etiological agent for cervical cancer, infects keratinocytes of the uterine cervix and has been shown to directly inhibit the IFN pathway. We evaluated IFN-κ, -β, and -γ gene expression in HPV-negative normal and HPV-positive pre-malignant and malignant ex vivo cervical tissue covering the entire spectrum of cervical disease. Quantitative real-time polymerase chain reaction and methods previously optimized for detecting low-expressing genes in cervical tissue were used. In contrast to IFN-β and -γ, IFN-κ mRNA prevalence and levels were unexpectedly higher in diseased compared with normal whole cervical tissue with highest levels observed in invasive carcinoma tissue. Strikingly, laser capture microdissection revealed an absence of IFN-κ mRNA in diseased epithelium, whereas stromal IFN-κ was found exclusively in diseased tissue. IFN-γ and IFN-β were likewise found to be upregulated in diseased cervical stroma. Immunofluorescence supports the involvement of monocytes and dendritic cells in the stromal induction of IFNs in diseased tissue. Further, using three-dimensional raft cultures in which the viral life cycle can be mimicked, human keratinocytes transfected with full-length HPV16 displayed a significant decrease in IFN-κ mRNA compared with non-transfected human keratinocytes. Altogether, these findings show that IFN-κ is down-regulated in cervical keratinocytes harboring HPV, which may be a contributing factor in the progression of a cervical lesion.