DNA methylation, the process by which a methyl group is added to a cytosine molecule, is essential for normal transcriptional regulation, in addition to X chromosome inactivation and genomic imprinting. Initial research has shown that exercise can both acutely (Barrès et al, 2012) and chronically (Nitert et al, 2012; Rönn et al, 2013) modify global and gene-specific levels of DNA methylation. DNA methyltransferases (DNMT) catalyse this process, however, there is a lack of literature concerning the specific molecular mechanisms by which exercise-induced epigenetic modifications occur. Interleukin 6 (IL-6) stimulation of cancer cell lines has been shown to augment DNA methyltransferase 1 (DNMT1) expression (Hodge et al, 2005), which suggests a possible pathway by which exercise is able to elicit changes in these epigenetic enzymes. Utilising 10 recreationally active males, the present study sought to elucidate the response of the de novo DNA methyltransferases 3A (DNMTA3) and 3B (DNMTB3) to 120 minutes of treadmill running at an intensity of 60% of individual velocity at VO2max (vVO2max), interspersed with 30 second sprints at 90% of vVO2max every 10 minutes – a protocol previously shown to elicit a transient increase in plasma IL-6 (Walshe et al, 2010). Peripheral blood mononuclear cells (PBMCs) were incubated with plasma isolated from exercising participants or recombinant IL-6 (rIL-6), followed by nuclear protein extraction and subsequent quantification of DNMT3A and DNMT3B concentrations. Paired samples T tests showed that nuclear concentrations of DNMT3B significantly decreased (p < 0.05) from 365.9 (± 363.6) ng∙mg protein-1 to 87.2 (± 73.3) ng∙mg protein-1 following the experimental protocol, with no change observed in DNMT3A. ‘High’ levels (100 ng∙ml-1) of rIL-6 stimulation resulted in significantly greater nuclear concentrations of both DNMT3A and DNMT3B, compared with ‘low’ concentrations (10 ng∙ml-1). This is the first known study to characterise the response of de novo methyltransferases to an acute bout of aerobic exercise or in vitro stimulation of PBMCs with rIL-6. The conflicting results suggest that IL-6 is not the only major regulator of DNMT nuclear transport, and that other plasma mediators may exert significant influence on the nuclear concentrations of these enzymes.