Myofibroblasts, the major effector cells in pathologic fibrosis, derive from the differentiation of fibroblasts driven by mediators such as transforming growth factor-β1 (TGF-β1) and biomechanical signals. Although the myofibroblast has traditionally been considered a terminally differentiated cell, the lipid mediator prostaglandin E₂ (PGE₂) has been shown to not only prevent but also reverse myofibroblast differentiation, as characterized by the ability of PGE₂ to diminish expression of collagen I and α-smooth muscle actin in established myofibroblasts. Here, we use microarrays to examine the extent of transcriptomic changes that occur during TGF-β1–induced differentiation and PGE₂-induced dedifferentiation of myofibroblasts. Normal primary human adult lung fibroblasts were cultured for 24 hours with or without TGF-β1 and treated for 48 hours with PGE₂. Gene expression levels were assessed from total RNA on the Affymetrix U219 microarray. TGF-β1 up-regulated 588 genes and down-regulated 689 genes compared with control cells. PGE₂ reversed the expression of 363 (62%) of the TGF-β1–up-regulated genes and 345 (50%) of the TGF-β1–down-regulated genes. Genes up-regulated by TGF-β1 and reversed by PGE₂ were enriched in annotations for Cell Adhesion, Contractile Fiber, and Actin Binding, whereas genes down-regulated by TGF-β1 but subsequently reversed by PGE₂ were enriched in annotations for Glycoprotein, Polysaccharide Binding, and Regulation of Cell Migration. Surprisingly, the genes whose expression was affected by PGE₂ differed between TGF-β1–induced myofibroblasts and undifferentiated fibroblasts. These data demonstrate the capacity of PGE₂ to effect marked global alterations in the transcriptomic program of differentiated myofibroblasts and emphasize the considerable plasticity of these cells.