DNA methyltransferase 3A (DNMT3A) is one of two human de novo DNA methyltransferases essential for transcription regulation during cellular development and differentiation. There is increasing evidence that RNA plays a role in directing DNA methylation to specific genomic locations within mammalian cells. Here, we describe two modes of RNA regulation of DNMT3A in vitro. We show a single-stranded RNA molecule that is antisense to the E-cadherin promoter binds tightly to the catalytic domain in a structurally dependent fashion causing potent inhibition of DNMT3A activity. Two other RNA molecules bind DNMT3A at an allosteric site outside the catalytic domain, causing no change in catalysis. Our observation of the potent and specific in vitro modulation of DNMT3A activity by RNA supports in vivo data that RNA interacts with DNMT3A to regulate transcription.