MicroRNA-21 (miR-21) upregulation, smad family member 7 (smad7) downregulation, epidermal growth factor (EGF) and transforming growth factor-β (TGF-β) actions contribute to breast cancer cell aggressiveness. However, their correlation and the relevant molecular mechanisms involved remain to be elucidated. The present study was undertaken to determine the association of miR-21, smad7, EGF and TGF-β with breast cancer cell invasion and migration and to identify the molecular mechanisms involved using immunohistochemistry and western blot analysis. In the present study, the plasma miR-21 levels were significantly increased in patients with breast cancer, as compared to the controls. Smad7 was confirmed to be a direct target of miR-21, by luciferase reporter and western blot assays. The downregulation of smad7 by miR-21 or sismad7 enhanced EGF-dependent invasion and migration, as well as TGF-β-dependent invasion and migration. The actions of miR-21 were abrogated by expressing a modified smad7 cDNA resistant to miR-21. Moreover, miR-21, EGF and TGF-β combined to markedly increase cancer cell invasion and migration, and this effect was blocked by the combination of erlotinib (an EGF receptor kinase inhibitor) and SB505124 (a type I TGF-β receptor inhibitor). A lower smad7 expression was identified in poorly differentiated breast cancers, as compared to well- to moderately differentiated breast cancers. Notably, antagonism of miR-21 decreased breast cancer cell proliferation and tumor growth in mouse models. In conclusion, our results demonstrated that plasma miR-21 levels may serve as a diagnostic marker in breast cancers, whereas miR-21 promotes breast cancer cell proliferation and invasion by suppressing smad7, which enhances EGF and TGF-β pathways.