DNA methylation profiling of TAFs reveals global demethylation and a selective impact on the TGF-beta pathway. Moreover, it suggests the fibrocyte origin of a fraction of TAFs, and identifies a novel prognostic biomarker in non-small cell lung cancer.Epigenetic changes through altered DNA methylation have been implicated in critical aspects of tumor progression, and have been extensively studied in a variety of cancer types. In contrast, our current knowledge of the aberrant genomic DNA methylation in tumor-associated fibroblasts (TAFs) or other stromal cells that act as critical coconspirators of tumor progression is very scarce. To address this gap of knowledge, we conducted genome-wide DNA methylation profiling on lung TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer patients using the HumanMethylation450 microarray. We found widespread DNA hypomethylation concomitant with focal gain of DNA methylation in TAFs compared to CFs. The aberrant DNA methylation landscape of TAFs had a global impact on gene expression and a selective impact on the TGF-beta pathway. The latter included promoter hypermethylation-associated SMAD3 silencing, which was associated with hyperresponsiveness to exogenous TGF-beta 1 in terms of contractility and extracellular matrix deposition. In turn, activation of CFs with exogenous TGF-beta 1 partially mimicked the epigenetic alterations observed in TAFs, suggesting that TGF-beta 1 may be necessary but not sufficient to elicit such alterations. Moreover, integrated pathway-enrichment analyses of the DNA methylation alterations revealed that a fraction of TAFs may be bone marrow-derived fibrocytes. Finally, survival analyses using DNA methylation and gene expression datasets identified aberrant DNA methylation on the EDARADD promoter sequence as a prognostic factor in non-small cell lung cancer patients. Our findings shed light on the unique origin and molecular alterations underlying the aberrant phenotype of lung TAFs, and identify a stromal biomarker with potential clinical relevance.