Abstract Gene silencing mediated by aberrant promoter DNA hypermethylation is one of the key features of cancer. Although such modification is heritable, its dynamic nature and reversibility through pharmacological interventions make it an attractive therapeutic target. Over the past few decades, various DNA methyltransferase inhibitors have been developed with the goal of reactivating aberrantly silenced genes. Two FDA-approved demthylating agents, decitabine and azacitidine are efficacious for the treatment of myeloid malignancies (MDS). It has been shown that transient exposure of tumor cells to low dose decitabine and azacitidine results in reduced tumorigenecity. However, the mechanism underlying clinical efficacies of these DNA methyltransferase inhibitors remains unclear. Here we investigate the long-term effect (up to 10 weeks) on population doubling time and the returning of DNA methylation (rebound methylation) after transient exposure to decitabine in three cancer cell lines, HCT116, T24 and HL-60. We show that transient exposure results in inhibition of cell growth and reduction of their colony formation capacity (up to 42 days). Furthermore, we also show that the rebound methylation occurs at different rates depending on the region and the type of genes. The discrepancy in population doubling time between the vehicle and decitabine treated cells disappears when rebound methylation fully restores, suggesting the presence of a correlation between cell doubling and rebound methylation. The majority of probes, which exhibit fast rebound methylation, are located in gene bodies. We show a positive correlation between gene body methylation and expression. However, there is a small group of fast rebound probes located at the promoters. Many of them belong to cancer-testis antigens, which get reactivated and quickly re-silenced. On the other hand, the majority of probes, which exhibit slow rebound methylation are located at promoters and show an inverse correlation between methylation and expression. The presence of a nucleosome-depleted region and enrichments of active histone marks, H3K4me3 and H2A.Z are observed at the promoters of those genes up to 42 days after drug treatment. Interesting, this group is enriched for genes down-regulated in colon adenocarcinoma when compared to normal colon (p=3.3e−6). Taken together, the sustained reactivation of those genes may be the driving force in reducing tumorigenecity of cancer cells. Our study elucidates the mechanism of decitabine, thus advancing our understanding of epigenetic therapy. Citation Format: Gangning Liang, Han Han, Daniel D. De Carvalho, Xiaojing Yang, Peter A. Jones. Transient exposure to decitabine results in sustained cell growth inhibition and long term DNA demethylation at specific loci. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 677. doi:10.1158/1538-7445.AM2013-677 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.