Myocardin, a cofactor of serum response factor (SRF), specifically induces the expression of contractile proteins to promote differentiation and contractile phenotype of smooth muscle cells (SMCs). SRF directly induces the transcription of microRNA-1 (miR-1) in cardiac and skeletal muscle precursor cells and miR-1 promotes the skeletal muscle differentiation and modulates cardiac hypertrophy. We aimed to examine whether miR-1 plays a role in the regulation of smooth muscle contractility. We found that miR-1 expression was induced by myocardin overexpression in human aortic SMCs. In a collagen lattice contraction assay using SMCs harboring a doxycycline-inducible expression system for myocardin, we found that myocardin expression increased the contractility of SMCs, which was significantly inhibited by exogenous miR-1. Our further studies revealed that exogenous miR-1, which did not affect myocardin or SRF expression, suppressed the expression of contractile proteins, such as alpha-SMA and SM22, and impaired the actin cytoskeletal organization. Taken together, our results have revealed that myocardin induces miR-1 expression, which represses the expression of contractile proteins and thereby inhibits the contractility of SMCs. Therefore, our findings suggest a role of miR-1 in the negative feedback loop in the regulation of contractility induced by myocardin.