Cancer growth is driven by cancer stem-like cells within a tumor, called cancer stem cells (CSCs). Since miRNAs can regulate cell-fate decisions, we compared miRNA expression in stem-like cells and differentiated cells from small cell lung cancer (SCLC) cell lines to develop further understanding of the molecular mechanisms involved in the pathogenesis of SCLC. First, SCLC stem-like cells were enriched by isolating sphere-forming cells using a defined serum-free medium. Further, microRNA microarrays were used to measure the expression of 1212 miRNAs in sphere-forming cells and parental cells. We found 86 miRNAs that were differentially expressed, including 48 upregulated miRNAs and 38 downregulated miRNAs between sphere-forming cells and parental cells. Among them, five downregulated miRNAs (let-7, miR-20, 21, 27a and 30b) and one upregulated miRNA (miR-149*) were selected for validation in 3 sets of SCLC cell lines by qRT-RCR. The qRT-PCR analysis confirmed that all six miRNAs were indeed differentially expressed. However, only miR-27a was consistently downregulated in sphere-forming cells of all 3 cell lines. Antagonizing miR-27a by inhibitor in parental cells enhanced proliferation, self renewal, and the proportion of undifferentiated cells in vitro. The candidate miRNA and some miRNAs with same seed sequence are predicted to have several target genes related to apoptosis, cell proliferation and cell cycle. Our results suggest that downregulation of miR-27a enhanced the stem-like properties of SCLC cells in vitro and may be critical to maintaining a stem cell function in SCLC.