The movement modes of epithelial cancer cells in three-dimensional (3D) environments include the mesenchymal mode, which is associated with local invasion, and the amoeboid mode, which facilitates distant metastasis. The migratory behavior of individual cancer cells is critical for tumor dissemination; however, the mechanism underlying regulation of the switch between movement modes is not clearly understood. For head and neck squamous cell carcinoma (HNSCC), local invasion is the major route of dissemination. We previously demonstrated that, in HNSCC cells, Twist1 represses let-7i expression to elicit mesenchymal-mode movement through activation of Ras-related C3 botulinum toxin substrate 1 (RAC1). In this study, we discover another important target gene of let-7i for regulating HNSCC migration. Using bioinformatic tools, we identified bone morphogenetic protein 4 (BMP4) as a candidate target of let-7i. Further experiments, including 3'-untranslated region (UTR) reporter assays, quantitative RT-PCR and western blotting, confirmed that BMP4 is a bona fide target repressed by let-7i. In the HNSCC cell line OECM-1, knockdown of BMP4 reduced mesenchymal-mode migration and invasion in 3D culture. In clinical HNSCC samples, let-7i expression was inversely correlated with BMP4 expression. Our results revealed that let-7i attenuates mesenchymal-mode migration of HNSCC cells through repression of a novel target, BMP4. (c) 2013 Elsevier Inc. All rights reserved.