Toll-like receptor3 (TLR3) has been confirmed to be differentially expressed in neuroblastoma (NB), and predicts a favorable prognosis with a high expression in tumor tissues. Treatment with TLR3 agonist--polyinosinic-polycytidylic acid [poly(I:C)] could induce significant but limited apoptosis in TLR3-expressing NB cells, suggesting that other viral RNA sensors, including melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) in the cytosolic compartment might also be implicated in poly(I:C)-induced NB cell death. MDA5 and RIG-I were induced by poly(I:C) to express in two of six NB cell lines, SK-N-AS (AS) and SK-N-FI, which were associated with up-regulation of caspase9 and active caspase3. While knockdown of either MDA5 or RIG-I alone is ineffective to decrease caspase9 and active caspase3, simultaneously targeting MDA5 and TLR3 showed the best effect to rescue poly(I:C) induced up-regulation of mitochondrial antiviral signaling protein (MAVS), caspase9, active caspase3, and apoptosis in AS cells. Over-expression of MDA5 in FaDu cells resulted in significantly less colony formation and more poly(I:C)-induced cell death. Further studies in human NB tissue samples revealed that MDA5 expression in NB tissues predicted a favorable prognosis synergistically with TLR3. Our findings indicate that MDA5 may serve as a complementary role in the TLR3 activated suppression of NB.