There is clear evidence that phenotypic modulation of smooth muscle cells (SMCs) contributes to the pathophysiology of vascular disease. Phenotypic modulation refers to the unique ability of SMCs to alter their phenotype in response to extracellular stimuli and is hallmarked by loss of SMC marker gene expression. The transcription factor KLF4 is a known powerful negative regulator of SMC marker gene expression that works, in part, by decreasing expression of the SRF co-factor, myocardin. KLF4 is not expressed in healthy adult SMCs but is increased in SMCs in response to vascular injury in vivo or PDGF-BB treatment in vitro. The aim of the present study was to determine the molecular mechanisms that regulate expression of KLF4 in phenotypically modulated SMCs. Results demonstrated that the transcription factor Sp1 regulated expression of KLF4 in SMC. The KLF4 promoter contains 3 consensus Sp1 binding sites. Using a series of truncated KLF4 promoters, we showed that only fragments containing these Sp1 sites could be activated by PDGF-BB. In addition, over-expression of Sp1 alone was sufficient to increase the activity of the KLF4 promoter. Moreover, inhibiting Sp1 expression with siRNA attenuated the effects of PDGF-BB on KLF4 expression. Mutation of the three Sp1 sites within the KLF4 promoter abolished both baseline and PDGF-BB-induced activity. Finally, results demonstrated enhanced Sp1 binding to the KLF4 promoter in SMCs treated with PDGF-BB in vitro and following vascular injury in vivo. Taken together, results suggest a novel role for Sp1 in increasing expression of KLF4 in phenotypically modulated SMCs.