Here we show that nitric oxide (NO) is a potent inhibitor of FTO demethylase activity by directly binding to the catalytic iron center, which causes global m6A hypermethylation of mRNA in cells and results in gene-specific enrichment of m6A on mRNA of NO-regulated transcripts. Both cell culture and tumor xenograft models demonstrated that endogenous NO synthesis can regulate m6A-mRNA levels and transcriptional changes of m6A-associated genes. These results build a direct link between NO and m6A-mRNA regulation and reveal a novel signaling mechanism of NO as an endogenous regulator of the epitranscriptome. Overall design: With the observation that NO directly inhibits FTO demethylase activity and globally increases m6A-mRNA, we sought to decipher whether this was functionally associated with transcriptional changes in NO-regulated genes. First, we quantified changes in transcription by performing RNA-sequencing on samples from two cell lines treated chronically (10 days) with NO.