C-type natriuretic peptide (CNP) possesses nitric oxide-like signaling mechanisms and actions in the vasculature, including the inhibition of fibrosis and vascular remodeling through counterregulation of transforming growth factor-β (TGF-β) signaling. The leucine zipper protein transforming growth factor stimulated clone 22 domain 1 (TSC22D1), cloned via its presumed binding to a GC-rich element in the CNP promoter, was the first protein to be described as a CNP transcription factor, but the lack of supporting evidence since its discovery and its lack of a classical DNA-binding site have left in question its role in the regulation of CNP by TGF-β and other factors. To define a specific role for TSC22D1 in CNP transcription, we have examined the effects of the profibrotic growth factors TGF-β1 and PDGF-BB on CNP mRNA expression in cultured human vascular smooth muscle cells (SMC) in which TSC22D1 expression was suppressed with small interfering RNA. Results showed that TGF-β and PDGF-BB significantly increased CNP expression in all three SMC types. Twenty-four-hour TGF-β-induced elevations in CNP were strongly correlated with changes in TSC22D1 mRNA levels, and both genes exhibited their greatest response to TGF-β1 in coronary artery SMC. Furthermore, siRNA suppression of TSC22D1 expression in coronary artery and aortic SMC by ∼90% resulted in 45-65% reductions of both PDGF- and TGF-β-stimulated CNP expression, respectively. These results support a postulated role of TSC22D1 as an enhancer of CNP transcription and suggest that TGF-β-induced upregulation of CNP expression in SMC may be mediated in part by increased transcription of TSC22D1.