Cervical cancer is one of the most common malignancies in women. MicroRNAs (miRNAs) are involved in a variety of fundamental cellular processes, including carcinogenesis. The potential utilization of aberrantly expressed miRNAs as novel biomarkers in cervical cancer diagnostics is growing. We investigated miRNA expression profiles during the progression of dysplasia in cervical epithelium to identify aberrantly expressed miRNAs. High-throughput miRNA profiling of high-grade precancerous lesions identified 79 miRNAs showing significant difference in expression values compared to normal cervical epithelium. Ten selected miRNAs were subsequently measured in an independent group of samples to validate them as promising biomarkers of cervical carcinogenesis. MicroRNAs miR-10b-5p, miR-34c-5p, miR-409-3p and miR-411-5p were confirmed as down regulated, while miR-10a-5p, miR-132-3p, miR-141-5p were significantly upregulated in dysplastic cervical tissues. Further investigation revealed an inverse correlation of miR-409-3p with E6 mRNA levels in pre-cancerous cervical lesions. Subsequent in vitro analyses showed a direct involvement of this miRNA in the regulation of E6 oncogene levels, thus confirming a potential tumor suppressor function of miR-409-3p in cervical malignancies. Hence, miR-409-3p may represent a useful early marker and a potential therapeutic target for cervical cancer.