Prostaglandin E₂ (PGE₂), via cAMP signaling, inhibits a variety of fibroblast functions relevant to fibrogenesis. Among these are their translation of collagen I protein and their differentiation to myofibroblasts. PKA is central to these actions, with cAMP binding to regulatory (R) subunits leading to the release of catalytic subunits. Here we examined the role of specific PKAR subunit isoforms in these inhibitory actions in transforming growth factor β-1 (TGFβ-1)-stimulated human lung fibroblasts (HLFs). HLFs expressed all four R subunit isoforms. siRNA-mediated knockdown of subunits PKARIα and PKARIIα had no effect on PGE₂ inhibition of either process. However, knockdown of PKARIβ selectively attenuated PGE₂ inhibition of collagen I protein expression, whereas knockdown of PKARIIβ selectively attenuated PGE₂ inhibition of expression of the myofibroblast differentiation marker, α-smooth muscle actin (α-SMA). cAMP analogs that selectively activate either PKARIβ or PKARIIβ exclusively inhibited collagen I synthesis or differentiation, respectively. In parallel, the PKARIβ agonist (but not a PKARIIβ agonist) reduced phosphorylation of two proteins involved in protein translation, protein kinase B (AKT) and mammalian target of rapamycin (mTOR). By contrast, the PKARIIβ agonist (but not a PKARIβ agonist) reduced levels of the differentiation-associated phosphorylated focal adhesion kinase (p-FAK) as well as the relative mRNA and protein expression of serum response factor (SRF), a transcription factor necessary for myofibroblast differentiation. Our results demonstrate that cAMP inhibition of collagen I translation and myofibroblast differentiation reflects the actions of distinct PKAR subunits.