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Background: Neuroblastoma (NB) is one of the most common malignant solid tumors to occur in children, characterized by a wide range of genetic and epigenetic aberrations. We studied whether modifications of the latter with a 5-aza-2′-deoxycytidine (decitabine, Dac) DNA methyltransferase inhibitor can provide a therapeutic advantage in NB. Methods: NB cells with or without <i>MYCN</i> amplification were treated with Dac. We used flow cytometry to measure cell apoptosis and death and mitochondrial reactive oxygen species (mtROS), microarray to analyze gene expression profile and bisulfite pyrosequencing to determine the methylation level of the <i>DDX58</i>/RIG-I promoter. Western blot was used to detect markers related to innate immune response and apoptotic signaling, while immunofluorescent imaging was used to determine dsRNA. We generated mtDNA depleted ρ⁰ cells using long-term exposure to low-dose ethidium bromide. Results: Dac preferentially induced a RIG-I-predominant innate immune response and cell apoptosis in SK-N-AS NB cells, significantly reduced the methylation level of the <i>DDX58</i>/RIG-I promoter and increased dsRNA accumulation in the cytosol. Dac down regulated mitochondrial genes related to redox homeostasis, but augmented mtROS production. ρ⁰ cells demonstrated a blunted response in innate immune response and apoptotic cell death, as well as greatly diminished dsRNA. The response of NB cells to CDDP and poly(I:C) was potentiated by Dac in association with increased mtROS, which was blunted in ρ⁰ cells. Conclusions: This study indicates that Dac effectively induces a RIG-I-related innate immune response and apoptotic signaling primarily in SK-N-AS NB cells by hypomethylating <i>DDX58</i>/RIG-I promoter, elevated mtROS and increased dsRNA. Dac can potentiate the cytotoxic effects of CDDP and poly(I:C) in NB cells.