A hallmark of dysfunctional fibroblast to myofibroblast differentiation associated with fibrotic disorders is persistent expression of ACTA2, the gene encoding the cyto-contractile protein smooth muscle α-actin. In this study, a PURB-specific gene knockdown approach was used in conjunction with biochemical analyses of protein subdomain structure and function to reveal the mechanism by which purine-rich element binding protein B (Purβ) restricts ACTA2 expression in mouse embryo fibroblasts (MEFs). Consistent with the hypothesized role of Purβ as a suppressor of myofibroblast differentiation, stable short hairpin RNA-mediated knockdown of Purβ in cultured MEFs promoted changes in cell morphology, actin isoform expression, and cell migration indicative of conversion to a myofibroblast-like phenotype. Promoter-reporter assays in transfected Purβ knockdown MEFs confirmed that these changes were attributable, in part, to derepression of ACTA2 transcription. To map the domains in Purβ responsible for ACTA2 repression, several recombinant truncation mutants were generated and analyzed based on hypothetical, computationally derived models of the tertiary and quaternary structure of Purβ. Discrete subdomains mediating sequence- and strand-specific cis-element binding, protein-protein interaction, and inhibition of a composite ACTA2 enhancer were identified using a combination of biochemical, biophysical, and cell-based assays. Our results indicate that the Purβ homodimer possesses three separate but unequal single-stranded DNA-binding modules formed by subdomain-specific inter- and intramolecular interactions. This structural arrangement suggests that the cooperative assembly of the dimeric Purβ repressor on the sense strand of the ACTA2 enhancer is dictated by the association of each subdomain with distinct purine-rich binding sites within the enhancer.