Transforming growth factor β-1(TGFβ1) plays a central role in the induction of myofibroblast differentiation and the development of pulmonary fibrosis. Cardiac glycosides (ouabain, digoxin) inhibit the Na+/K+-ATPase and thus increase the intracellular [Na+]/[K+] ratio within cells. Previous microarray analysis showed cyclooxygenase 2 (COX-2), the rate limiting enzyme required for the synthesis of prostaglandins, was upregulated upon ouabain treatment in multiple cell lines. Given the anti-fibrotic effects of prostaglandins through the activation of protein kinase A (PKA), we examined if cardiac glycosides stimulate COX-2 expression in human lung fibroblasts (HLF) and how they affect myofibroblast differentiation. Ouabain dramatically upregulated COX-2 expression in HLF and induced a sustained activation of PKA, which was inhibited with NS-398 (COX-2 inhibitor) and COX-2 knockdown. Ouabain induced COX-2 expression was lost with treatment of KB-R4943, a Na+/Ca2+ exchanger inhibitor. Furthermore, ouabain inhibited TGFβ1 stimulated Rho activation, stress fiber formation, SRF activation and myofibroblast differentiation (measured by expression of smooth muscle α-actin, collagen-1, and fibronectin), which were tightly coupled with the change in the intracellular [Na+]/[K+] ratio. Although the expression of COX-2 and activation of PKA were highly associated with these effects, neither the inhibition of COX-2 enzymatic activity by NS-398 nor the inhibition of COX-2 expression by siRNA or KB-R4943, rescued the effects of ouabain on TGFβ1 stimulated myofibroblast activation. ,When looking directly at the canonical TGFβ1 signaling pathway, we found the inhibition of the Na+/K+-ATPase by K+-free media/ouabain, resulted in a dramatic downregulation of TGFβR2 mRNA and protein. The downregulation of TGFβR2 was accompanied with the inhibition of TGFβ1-induced SMAD2 phosphorylation and myofibroblast differentiation. Given the essential role of TGFβR2 in the initiation TGFβ1-induced signaling and the fibrotic response, we tested if overexpression of TGFβR2 could reverse the effects of ouabain on myofibroblast activation. Overexpression of TGFβR2, by multiple mechanisms, was unable to abolish the inhibitory actions of ouabain on myofibroblast differentiation. Nonetheless, the impedance of the Na+/K+-ATPase activity by ouabain dramatically suppressed TGFβ1-induced myofibroblast differentiation at nanomolar concentrations, as a result we investigated if ouabain exhibited anti-fibrotic properties in vivo, using the bleomycin model of pulmonary fibrosis in cardiac glycoside sensitive mice (α1S/S mice). To confirm the sensitivity to cardiac glycosides in these mutant mice, we examined the effects of ouabain on both wild-type and α1S/S fibroblasts. Isolated wild-type mouse lung fibroblasts showed no change in TGFβR2 mRNA levels after ouabain treatment, while α1S/S mouse lung fibroblasts showed a drastic downregulation of TGFβR2 mRNA in the presence of ouabain, further indicating the inhibition of the Na+/K+-ATPase is required for the downregulation of TGFβR2. Moreover, mice treated with 50 µg/kg per day of ouabain after bleomycin injury, demonstrated decreased collagen deposition in the lung as compared to injured PBS controls via the hydroyproline assay. ,Together, these data show that ouabain, through the increase in intracellular [Na+]/[K+] ratio, drives the induction of COX-2 expression and PKA activation, which is accompanied by decreased Rho activation and myofibroblast differentiation in response to TGFβ1. Furthermore, nanomolar concentrations of ouabain profoundly downregulated TGFβR2, an important player in the initiation of TGFβ1 signaling and the fibrotic response. However, COX-2 expression, PKA activation, and downregulation of TGFβR2 were not sufficient for inhibition of the fibrotic effects of TGFβ1 by ouabain, suggesting additional mechanisms must exist. However, we have demonstrated ouabain potently inhibited TGFβ1 induced myofibroblast differentiation and attenuated pulmonary fibrosis in the bleomycin model, indicating an important role of the Na+/K+-ATPase in fibrogenesis in vitro and in vivo.