Abstract Ovarian cancer ranks as the most deadly gynecologic cancer, and there is an urgent need to develop more effective therapies. Previous studies have shown that G9A, a histone methyltransferase that catalyzes mono- and dimethylation of histone H3 lysine9, is highly expressed in ovarian cancer tumors, and its overexpression is associated with poor prognosis. Here we report that pharmacologic inhibition of G9A in ovarian cancer cell lines with high levels of G9A expression induces synergistic antitumor effects when combined with the DNA methylation inhibitor (DNMTi) 5-aza-2′-deoxycytidine (5-aza-CdR). These antitumor effects included upregulation of endogenous retroviruses (ERV), activation of the viral defense response, and induction of cell death, which have been termed "viral mimicry" effects induced by DNMTi. G9Ai treatment further reduced H3K9me2 levels within the long terminal repeat regions of ERV, resulting in further increases of ERV expression and enhancing "viral mimicry" effects. In contrast, G9Ai and 5-aza-CdR were not synergistic in cell lines with low basal G9A levels. Taken together, our results suggest that the synergistic effects of combination treatment with DNMTi and G9Ai may serve as a novel therapeutic strategy for patients with ovarian cancer with high levels of G9A expression. Significance: Dual inhibition of DNA methylation and histone H3 lysine 9 dimethylation by 5-aza-CdR and G9Ai results in synergistic upregulation of ERV and induces an antiviral response, serving as a basis for exploring this novel combination treatment in patients with ovarian cancer. Cancer Res; 78(20); 5754–66. ©2018 AACR.