The role of the 3'untranslated region (UTR) of the dengue virus (DENV) genome during viral translation remains to be elucidated. We assessed the contribution of well-defined RNA elements in the 3'UTR of DENV-2 to viral translation using a virus-induced reporting gene system and deoxyribozymes (DRzs) targeting the 3'UTR of the DENV-2 genome. Results show that mRNAs carrying a deletion of repeated conserved sequence (RCS2)-CS2 are translated less efficiently than wild type mRNAs. However, mRNAs with a deletion of CS1-stem loop (SL) are translated more efficiently. Thus, CS1-SL and RCS2-CS2 may have different effects on translational regulation. Additionally, the translation-suppressing effect of CS1-SL or the SL element is further confirmed in DENV-2-infected cells using DRzs. Mutagenesis studies show that, rather than the secondary structure, nucleotides 10663-10677 and 10709-10723 are responsible for translational suppression of SL. Overall, our results demonstrate that sequences and elements within the DENV-2 3'UTR regulate viral translation.