The cancer–testis/cancer germline antigen, NY-ESO-1, is a vaccine target in epithelial ovarian cancer (EOC), but its limited expression is a barrier to vaccine efficacy. As NY-ESO-1 is regulated by DNA methylation, we hypothesized that DNA methyltransferase inhibitors may augment NY-ESO-1 vaccine therapy. In agreement, global DNA hypomethylation in EOC was associated with the presence of circulating antibodies to NY-ESO-1. Preclinical studies using EOC cell lines showed that decitabine treatment enhanced both NY-ESO-1 expression and NY-ESO-1–specific CTL-mediated responses. On the basis of these observations, we performed a phase I dose-escalation trial of decitabine, as an addition to NY-ESO-1 vaccine and doxorubicin liposome chemotherapy, in 12 patients with relapsed EOC. The regimen was safe, with limited and clinically manageable toxicities. Both global and promoter-specific DNA hypomethylation occurred in blood and circulating DNAs, the latter of which may reflect tumor cell responses. Increased NY-ESO-1 serum antibodies and T-cell responses were observed in the majority of patients, and antibody spreading to additional tumor antigens was also observed. Finally, disease stabilization or partial clinical response occurred in six of ten evaluable patients. On the basis of these encouraging results, evaluation of similar combinatorial chemo-immunotherapy regimens in EOC and other tumor types is warranted. Cancer Immunol Res; 2(1); 37–49. ©2014 AACR.