The serum response factor (SRF) regulates the transcription of target genes by binding to serum response elements in dimeric form and by interacting with ternary complex factors. In this study, we have analyzed the role of the serum response factor and mechanisms that regulate its activity in tumor progression utilizing a multistage model of mouse skin carcinogenesis. We demonstrate elevated SRF DNA binding activity only in the cell lines that have undergone an epithelial to mesenchymal transition and have increased actin stress fiber formation. Transient transfection experiments of activated or dominant negative forms of RhoA showed that the high activity of SRF and the induced formation of actin stress fibers in cells with spindle morphology were mediated by RhoA signaling. A dominant negative form of SRF inhibited RhoA-induced actin polymerization and stress fiber formation. The DNA binding activity of SRF in mesenchymal tumor cells was also correlated with elevated expression of SRF target genes, similar to SRF itself, actin, and vinculin. These observations suggest for the first time that SRF may play an important role in tumor progression, specifically at the transition to an invasive metastatic stage of carcinogenesis.