Short non-coding RNAs, called microRNAs (miRNAs), regulate cell biology by affecting the expression of target genes. However, we know little about the miRNAs regulating the growth and progression of Ewing's sarcoma (ES). To identify possible oncogenic factors in ES, we used a microarray-based approach to profile the changes in the expression of miRNAs and the downstream mRNAs in five ES cell lines. One miRNA, miR-138, was significantly downregulated, whereas the expression of focal adhesion kinase (FAK) was significantly upregulated in all tested ES cells. When miR-138 was transfected into ES cell lines, the expression of FAK in these cells was greatly suppressed and inhibited the proliferation and mobility of ES cells. Overexpression of miR-138 in vitro resulted in further inhibition of the cell cycle at the G1 phase and in the induction of anoikis, in a dose-and time-dependent manner. Moreover, miR-138 overexpression in ES cells significantly suppressed the number of distant metastases in vivo. The data in the present study demonstrates for the first time a novel mechanism that regulates the expression of FAK via miR-138 in ES cells.