Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Cytokines such as transforming growth factor-β (TGF-β) play a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and other profibrotic responses, but less is known about pathways that might inhibit fibrosis. Increased cAMP formation inhibits myofibroblast differentiation and collagen production by cardiac fibroblasts, but the mechanism of this inhibition is not known. We sought to characterize the signaling pathways by which cAMP-elevating agents alter collagen expression and myofibroblast differentiation. Treatment with 10 μM forskolin or isoproterenol increased cAMP production and cAMP response element binding protein (CREB) phosphorylation in cardiac fibroblasts and inhibited serum- or TGF-β-stimulated collagen synthesis by 37% or more. These same cAMP-elevating agents blunted TGF-β-stimulated expression of collagen I, collagen III, and α-smooth muscle actin. Forskolin or isoproterenol treatment blocked the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by TGF-β despite the fact that these cAMP-elevating agents stimulated ERK1/2 activation on their own. cAMP-elevating agents also attenuated the activation of c-Jun NH₂-terminal kinase and reduced binding of the transcriptional coactivator CREB-binding protein 1 to transcriptional complexes containing Smad2, Smad3, and Smad4. Pharmacological inhibition of ERK completely blocked TGF-β-stimulated collagen gene expression, but expression of an active mutant of MEK was additive with TGF-β treatment. Thus, cAMP-elevating agents inhibit the profibrotic effects of TGF-β in cardiac fibroblasts largely through inhibiting ERK1/2 phosphorylation but also by reducing Smad-mediated recruitment of transcriptional coactivators.