microRNAs are small endogenous noncoding RNAs, which post-transcriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. To investigate the potential functional relationship of miR-10b and syndecan-1, MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behavior was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. miR-10b overexpression induced post-transcriptional downregulation of syndecan-1, as demonstrated by quantitative real-time PCR (qPCR), flow cytometry, and 3′UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation was increased. Syndecan-1 coimmunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1-deficient cells revealed upregulation of ATF-2, COX-2, cadherin-11, vinculin, actin γ 2, MYL9, transgelin-1, RhoA/C, matrix metalloproteinase 2 (MMP2) and heparanase, and downregulation of AML1/RUNX1, E-cadherin, CLDN1, p21WAF/CIP, cyclin-dependent kinase 6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase-dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.