Methyltransferase inhibitors commonly used in clinical trials promote tumor cell death, but their detailed cytotoxic action is not yet fully understood. A deeper knowledge about their apotosis-inducing mechanisms and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b might allow the design of more effective drugs with lower cytotoxicity. 5-aza-cytidine (5-aza-CR), a potent inhibitor of DNMT1, is known to induce demethylation and reactivation of silenced genes. In this study, we investigated the p53 dependence of apoptotic, cell cycle, and growth inhibitory effects of 5-aza-CR, as well as the influence on the expression level of DNMT1, DNMT3a, and DNMT3b in the colon cancer cell line HCT-116. Exposure to 5-aza-CR induced the up-regulation of genes promoting cell cycle arrest and DNA repair (p21(WAF1) and GADD45) or apoptosis (p53, RIPK2, Bak1, caspase 5, and caspase 6). In parallel, there was a down-regulation of antiapoptotic Bcl2 protein and the G(2)/M-mediator cyclin B1. Co-incubation with pifithrin-alpha (PFT-alpha), a selective p53 inhibitor, restored GADD45, Bcl2, cyclin B1, and p21(WAF1) expression levels and almost completely reversed the growth inhibitory, cell cycle, and apoptotic effects of 5-aza-CR. 5-aza-CR treatment caused global demethylation and reactivation of p16(INK4) expression. There was a marked decrease in DNMT1 and DNMT3a mRNA expression, with PFT-alpha reversing these effects. However, 5-aza-CR treatment did not modulate DNMT3b expression. Our data demonstrate that 5-aza-CR action in HCT-116 is mediated by p53 and its downstream effectors p21(WAF1) and GADD45. This is the first report to show a link between p53 and regulation of DNMT1 and de novo methyltransferase DNMT3a.