Stretch of the vascular wall promotes smooth muscle contractile differentiation. This effect requires L-type calcium influx, Rho-activation and actin polymerization. A novel mechanism for the regulation of smooth muscle phenotype has been identified by the discovery of smooth muscle specific microRNAs (miRNAs). miRNAs are short, noncoding RNAs estimated to play a fundamental role in a number of human disease states. To delete smooth muscle miRNAs we used a cell specific and tamoxifen-inducible Dicer KO mouse. Vascular mechanosensing was then investigated in control and Dicer KO vessels by subjecting portal veins to mechanical stretch in organ culture. Using this method, stretch promotes calcium- dependent Rho-activation and contractile differentiation of vascular smooth muscle in control mice. However, in the absence of miRNAs we found that the stretch response was reduced or completely ablated. In addition, we found that loss of miRNAs resulted in reduced expression of L-type calcium channels, which are crucial for stretch-induced contractile differentiation in smooth muscle. On the other hand, acute stretch- sensitive growth signaling, which is independent of influx through L-type calcium channels, was not affected by Dicer KO. Furthermore, inhibition of miR-145, a highly expressed miRNA in smooth muscle, resulted in a similar reduction of L-type calcium channel expression as Dicer KO. This effect was abolished by the CaMKII inhibitor KN93, suggesting that activation of CamKIId, a target of miR-145, plays a role in the down regulation of L- type channel expression. In support of this idea, we found that the expression of CamKIId was up- regulated in Dicer KO portal veins. In summary, our results suggest that miRNAs play a crucial role for stretch-induced contractile differentiation in the vascular wall, in part via miR-145 and CamKIId dependent regulation of L-type calcium channels.