Myofibroblasts are pathogenic in pulmonary fibrotic disease due to their exuberant production of matrix rich in collagen that interferes with gas exchange and the ability of these cells to contract and distort the alveolar space. Transforming growth factor-β1 (TGF-β1) is a well-known inducer of myofibroblast differentiation. TGF-β1-induced transformation of fibroblasts to apoptosis-resistant myofibroblasts is adhesion-dependent and focal adhesion kinase (FAK)-mediated. Prostaglandin E₂ (PGE₂) inhibits this differentiation via E prostanoid receptor 2 (EP2) signaling and cAMP elevation, but whether PGE₂ does so by interfering with TGF-β1 signaling is unknown. Thus we examined the effects of PGE₂ in the presence and absence of TGF-β1 stimulation on candidate signaling pathways in human lung fibroblasts. We now demonstrate that PGE₂ does not interfere with TGF-β1-induced Smad phosphorylation or its translocation to the nucleus. Rather, PGE₂ has dramatic effects on cell shape and cytoskeletal architecture and disrupts the formation of appropriate focal adhesions. PGE₂ treatment diminishes TGF-β1-induced phosphorylation of paxillin, STAT-3, and FAK and, in turn, limits activation of the protein kinase B (PKB/Akt) pathway. These alterations do not, however, result in increased apoptosis within the first 24 h of treatment. Interestingly, the effects of PGE₂ stimulation alone do not always mirror the effects of PGE₂ in the presence of TGF-β1, indicating that the context for EP2 signaling is different in the presence of TGF-β1. Taken together, our results demonstrate that PGE₂ has the potential to limit TGF-β1-induced myofibroblast differentiation via adhesion-dependent, but Smad-independent, pathways.