Small cell lung cancer (SCLC) constitutes approximately 15% of all diagnosed lung cancers. SCLC is a particularly lethal malignancy, as the 2-year survival rate after appropriate treatment is less than 5%. The patients with SCLC have not been received a benefit of the recently developed molecular targeted treatment. Therefore, a new treatment strategy is necessary for the patients. The molecular mechanisms underlying the aggressiveness of SCLC cells and their development of treatment-resistance are still ambiguous. In this study, we newly constructed a microRNA (miRNA) expression signature of SCLC by analysis of autopsy specimens. Based on the resultant signature, four miRNAs (miR-27a-5p, miR-485-3p, miR-34-5p and miR-574-3p) were found to be candidate anti-tumor miRNAs. To investigate their functional importance, we first validated the downregulation of miR-27a-5p and miR-34b-3p in SCLC clinical specimens. Next, we demonstrated that ectopic expression of both miR-27a-5p and miR-34b-3p significantly inhibited cancer cell aggressiveness. Our in silico analyses showed that four genes (topoisomerase 2 alpha (TOP2A), maternal embryonic leucine zipper kinase (MELK), centromere protein F (CENPF) and SRY-box 1 (SOX1) were identified as miR-27a-5p- and miR-34b-3p-regulated genes. Based on immunohistochemical analysis, TOP2A, MELK and CENPF were involved in SCLC pathogenesis. These genes might contribute to high proliferation and early metastatic spread of SCLC cells. Elucidation of differentially expressed miRNA-mediated cancer pathways based on SCLC signature may provide new insights into the mechanisms of SCLC pathogenesis.