Objective The importance of miRNAs in development and cell biology has previously been dissected by Dicer gene disruption in mice. However, this approach has not been used to investigate the role of miRNAs specifically in smooth muscle (SM). Thus, the aim of this study was to investigate the role of global miRNA knockdown by deletion of Dicer in vascular SM (SM Dicer KO) for vascular function and development. Methods Inactivation of Dicer in smooth muscle was achieved by cross-breeding SM22Cre transgenic mice or inducible MHC-ERT2Cre mice with Dicer floxed mice. Results Constitutive SM22-targeted deletion of Dicer during development results in embryonic lethality at E16.5, associated with widespread internal hemorrhage. The aorta of SM Dicer KO embryos exhibited an increased lumen diameter, reduced smooth muscle cell proliferation and decreased wall thickness. Electron microscopy analysis revealed polygonal and poorly organized smooth muscle cells with reduced myofilament content in the aortic wall of SM Dicer KO embryos. Active length-tension relationships in response to 80mM KCl revealed a decrease in contractile force in SM Dicer KO umbilical arteries. Using western blot analysis of umbilical cords at E16.5 we show that Dicer KO smooth muscle have a decreased expression of SMC specific proteins in addition to a loss of Dicer expression. Several of our findings in adult inducible SM Dicer KO mice were consistent with the embryonic phenotype. In isolated smooth muscle cells from Dicer KO aorta, loss of contractile protein markers was associated with a reduction in actin stress fiber formation. Transduction of Dicer KO SMC with Ad.Myocd or transfection of Mir- 145 mimic rescued the loss of SMC specific gene expression in these cells. Conclusion miRNAs play a crucial role in vascular SM development and function by regulation of proliferation and contractile differentiation, possibly via altered actin dynamics. This model of SM specific global miRNA knockdown is of great value for future identification of specific miRNAs that regulate SM phenotype.