MicroRNA 143/145 (miR143/145) is restricted to adult smooth muscle cell (SMC) lineages and mediates, in part, the expression of several SMC contractile genes. Although the function of miR143/145 has begun to be elucidated, its transcriptional regulation in response to various signaling inputs is poorly understood. In an effort to define a miR signature for SMC differentiation, we screened human coronary artery SMCs for miRs modulated by TGF-β1, a known stimulus for SMC differentiation. Array analysis revealed a number of TGF-β1-induced miRs, including miR143/145. Validation studies showed that TGF-β1 stimulated miR143/145 expression in a dose- and time-dependent manner. We utilized several chemical inhibitors and found that SB203580, a specific inhibitor of p38MAPK, significantly decreased TGF-β1-induced miR143/145 expression. siRNA studies demonstrated that the effect of TGF-β1 on miR143/145 was dependent upon the myocardin and serum response factor transcriptional switch as well as SMAD4. TGF-β1 stimulated a 580-bp human miR143/145 enhancer, and mutagenesis studies revealed a critical role for both a known CArG box and an adjacent SMAD-binding element for full TGF-β1-dependent activation of the enhancer. Chromatin immunoprecipitation assays documented TGF-β1-mediated enrichment of SMAD3 and SMAD4 binding over the enhancer region containing the SMAD-binding element. Pre-miR145 strongly promoted SMC differentiation, whereas an anti-miR145 partially blocked TGF-β1-induced SMC differentiation. These results demonstrate a dual pathway for TGF-β1-induced transcription of miR143/145, thus revealing a novel mechanism underlying TGF-β1-induced human vascular SMC differentiation.