Transforming growth factor-β (TGF-β) is a cytokine implicated in wound healing and in the pathogenesis of pulmonary fibrosis. TGF-β stimulates myofibroblast differentiation characterized by expression of contractile smooth muscle (SM)-specific proteins such as SM–α-actin. In the present study, we examined the role of serum response factor (SRF) in the mechanism of TGF-β–induced pulmonary myofibroblast differentiation of human lung fibroblasts (HLF). TGF-β stimulated SM–α-actin expression in HLF, which paralleled with a profound induction of SRF expression and activity. Inhibition of SRF by the pharmacologic SRF inhibitor (CCG-1423), or via adenovirus-mediated transduction of SRF short hairpin RNA (shSRF), blocked the expression of both SRF and SM–α-actin in response to TGF-β without affecting Smad-mediated signaling of TGF-β. However, forced expression of SRF on its own did not promote SM–α-actin expression, whereas expression of the constitutively transactivated SRF fusion protein (SRF-VP16) was sufficient to induce SM–α-actin expression, suggesting that both expression and transactivation of SRF are important. Activation of protein kinase A (PKA) by forskolin or iloprost resulted in a significant inhibition of SM–α-actin expression induced by TGF-β, and this was associated with inhibition of both SRF expression and activity, but not of Smad-mediated gene transcription. In summary, this is the first direct demonstration that TGF-β–induced pulmonary myofibroblast differentiation is mediated by SRF, and that inhibition of myofibroblast differentiation by PKA occurs through down-regulation of SRF expression levels and SRF activity, independent of Smad signaling.