Abstract Background: MicroRNAs (miRNAs), a class of small non-coding RNAs, regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. A growing body of evidence suggests that miRNAs contribute to cervical squamous cell carcinoma (cervical-SCC) progression, development, and metastasis. Recent our miRNA expression signature of SCC (hypopharyngeal-SCC and esophageal-SCC) revealed that microRNA-218 (miR-218) was significantly reduced in cancer tissues. The study of the aim was to investigate the functional significance of miR-218 and its mediated molecular pathways in cervical-SCC. Methods: Gain-of-function studies were performed to investigate cancer cell proliferation, migration and invasion by restoration of mature miRNAs into HPV positive or negative cervical-SCC cell lines (CaSKi, HeLa, ME180 and YUMOTO). To identify the biological processes or pathways potentially regulated by the miRNAs, we applied genome-wide gene expression analysis and in silico study. The GENECODIS software assigned a number of the putative miRNA targets to known pathways in KEGG [http://www.genome.jp/kegg/pathway.html]. Gene expression analyses of all candidate genes involved in each of the pathways using GEO (http://www.ncbi.nlm.nih.gov/geo/) database. Results: Expression levels of miR-218 were significantly reduced in cervical-SCC clinical specimens compared to adjacent non-cancerous tissues (P<0.0001). Restoration of miR-218 significantly inhibited cancer cell migration and invasion in all cervical-SCC cell lines. These data indicated that miR-218 act as a tumor suppressor in cervical-SCC. Our in silico analysis showed that miR-218 appeared to be an important modulator of tumor cell processes through suppression of many targets, particularly those involved in “focal adhesion” signaling pathways (P<0.05). Gene expression data indicated that LAMB3 and LAMC1 were up-regulated in cervical-SCC clinical specimens. The laminins are an important and biologically active part of the basal lamina, the function of that are various such as influencing cell differentiation, migration and adhesion as well as proliferation and cell survival. Conclusions: Aberrant expression of miRNAs has shed light on recent miRNA signatures and identification of tumor suppressive miRNA mediated novel molecular pathways in cervical-SCC. Tumor suppressive miR-218-mediated novel molecular pathways provide a new insight of cervical-SCC oncogenesis and metastasis. Citation Format: Noriko Yamamoto, Takashi Kinosita, Nijiro Nohata, Toshihiko Idesako, Hirofumi Yoshino, Hideki Enokida, Masayuki Nakagawa, Makio Syozu, Naohiko Seki. Tumor suppressive microRNA-218 mediated focal adhesion pathways in cervical squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4171. doi:10.1158/1538-7445.AM2013-4171