Multiple myeloma (MM) is an incurable plasma cell neoplasm despite the recent introduction of novel therapies such as bortezomib and immunomodulatory drugs. Considering the heavily disrupted epigenetic landscape in cancer and MM, an alternative approach is the use of epigenetic-modifying drugs. Here we investigated the preclinical anti-myeloma activity of decitabine (DAC), a demethylating agent with validated use in myelodysplastic syndromes. In our study, DAC inhibited the proliferation of both murine 5T33MMvt cells (IC50 = 101 nM at 48 hours) and human myeloma cell lines (IC50 of 750 nM for the RPMI-8226 and 5 µM for the OPM-2 at 48 hours, respectively), induced caspase-dependent apoptosis and modulated the expression of Bcl-2 family members. DAC also triggered a DNA damage response, cell cycle arrest, and modulated the expression of several cell cycle regulator proteins. Moreover, DAC-induced cytotoxicity seemed to be independent of the transcriptional activity of p53. We next confirmed the anti-myeloma properties of DAC in vivo, using the 5T33MM murine model. Tumour load was significantly reduced and overall survival significantly prolonged in mice treated with decitabine. We conclude that DAC has potent anti-myeloma activity in vitro and in vivo in the 5T33MM murine model probably by inducing p53-independent cytotoxicity. Conversely, the in vitro effects of DAC on human MM cell lines were modest, encouraging the use of DAC in combination with other epigenetic-modulating agents.