Abstract Background DNA methylation is important for the maintenance of the silent state of genes on the inactive X chromosome (Xi). Here, we screened for siRNAs and chemicals that reactivate an Xi-linked reporter in the presence of 5-aza-2′-deoxycytidine (5-aza-2′-dC), an inhibitor of DNA methyltransferase 1, at a concentration that, on its own, is not sufficient for Xi-reactivation. Results We found that inhibition of ribonucleotide reductase (RNR) induced expression of the reporter. RNR inhibition potentiated the effect of 5-aza-2′-dC by enhancing its DNA incorporation, thereby decreasing DNA methylation levels genome-wide. Since both 5-aza-2′-dC and RNR-inhibitors are used in the treatment of hematological malignancies, we treated myeloid leukemia cell lines with 5-aza-2′-dC and the RNR-inhibitor hydroxyurea, and observed synergistic inhibition of cell growth and a decrease in genome-wide DNA methylation. Conclusions Taken together, our study identifies a drug combination that enhances DNA demethylation by altering nucleotide metabolism. This demonstrates that Xi-reactivation assays can be used to optimize the epigenetic activity of drug combinations.