Two genes (MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT). MAT1A is silenced in hepatocellular carcinoma (HCC), and absence of MAT1A leads to spontaneous development of HCC in mice. Previous report correlated promoter methylation to silencing of MAT1A but definitive proof was lacking. Here we investigated the role of methylation in regulating MAT1A expression. There are three MspI/HpaII sites from −1,913 to +160 of the human MAT1A gene (numbered relative to the translational start site) at position −977, +10, and +88. Bisulfite treatment and DNA sequencing, and Southern blot analysis showed that methylation at +10 and +88, but not −977, correlated with lack of MAT1A expression. MAT1A promoter construct methylated at −977, +10 or +88 position has 0.7‐fold, 3‐fold, and 1.6‐fold lower promoter activity, respectively. Methylation at −977 and +10 did not inhibit the promoter more than methylation at +10 alone; while methylation at +10 and +88 reduced promoter activity by 60%. Mutation of +10 and +88 sites also resulted in 40% reduction of promoter activity. Reactivation of MAT1A correlated with demethylation of +10 and +88. In vitro transcription assay showed that methylation or mutation of +10 and +88 sites reduced transcription. In conclusion, our data support the novel finding that methylation of the MAT1A coding region can inhibit gene transcription. This represents a key mechanism for decreased MAT1A expression in HCC and a target for therapy. To our knowledge, this is the first example of coding region methylation inhibiting transcription of a mammalian gene. J. Cell. Physiol. 227: 1583–1591, 2012. © 2011 Wiley Periodicals, Inc.