Glaucoma is an optic neuropathy that affects 60 million people worldwide. There is an underlying fibrosis associated with the lamina cribrosa (LC) in glaucoma. DNA methylation is well established in regulating fibrosis and may be a therapeutic target for glaucoma. The purpose of this study was to compare global DNA methylation levels in primary human normal (NLC) and glaucomatous (GLC) cells, and to investigate DNA methylation in driving fibrosis through regulation of transforming growth factor β1 (TGFβ1). LC cells were cultured from normal and glaucomatous human donors. Global methylation was assessed by ELISA. qPCR was conducted for DNA methyltransferases (DNMTs), methyl-CpG-binding protein 2 (MeCP2), TGFβ 1 and 2, collagen 1α1 (COL1A1), and α-smooth muscle actin (αSMA). TGFβ1 and DNMT1 were examined by immunofluorescence. Methylation of the TGFβ1 promoter was determined by methylation-specific PCR (MSP). Global DNA methylation demonstrated an increase in GLC compared with NLC cells (P<0.05). The previously mentioned methylation and matrix genes were increased in GLC compared with NLC cells (P<0.05). Immunofluorescence showed increased TGFβ1 and DNMT1 in GLC compared with NLC cells. MSP showed increased unmethylated DNA in the TGFβ1 promoter of GLC compared with NLC cells. We found increased expression of fibrotic genes in GLC cells and demonstrated an increase in global DNA methylation and in associated enzymes in GLC cells. Furthermore, we showed decreased promoter methylation of TGFβ1 in GLC cells. Determining a role for methylation in glaucoma and in regulating TGFβ1 may provide a novel therapeutic approach.