Dengue (DENV) is the most important mosquito-borne virus affecting humans and is transmitted by Aedes aegypti and Ae. albopictus mosquitoes. DENV is an enveloped virus in the family Flaviviridae, with a positive-strand RNA genome that contains a single open reading frame, which is flanked by highly structured 5' and 3' untranslated regions (UTRs). Thus far, investigations of cis-acting regulatory RNA elements have focused on those within the UTRs, though we have recently described an RNA element in the DENV coding-region, the capsid-coding hairpin, which has been shown to regulate both viral translation and RNA synthesis. Using sequence alignments and secondary structure prediction algorithms, additional coding-region RNA elements were identified and characterized for their ability to modulate the viral life cycle in mammalian and mosquito cells. Three candidate RNA structure elements were identified in the capsid- and NS5-coding region, termed capsid-1 (C-1), C-2 and NS5-2. Three RNA sequence elements were identified in the capsid-, prM- and NS5-coding region of the genome, termed the prM-coding conserved region 1 (prMCR1), the NS5-coding conserved region 1 (NS5CR1) and the conserved capsid-coding region 1 (CCR1). Mutations were introduced into a DENV2 infectious clone to disrupt either the conserved predicted secondary structures or the primary nucleotide sequence while maintaining reading frame, amino acid R groups, adjacent predicted secondary structures, and codon usage bias in both baby hamster kidney (BHK) and Ae. albopictus mosquito (C6/36) cells. Initial infectivity screens showed that neither C-1, C-2, NS5-2, prMCR1 nor NS5CR1 were critical for the viral life cycle. However, changes to the primary nucleotide sequence of CCR1 were shown to decrease viral titer in both BHK and C6/36 cells, though its effects were shown to have a more critical role in mosquito cells. The defects in viral replication in C6/36 cells were confirmed in vivo, where mutations to CCR1 decrease viral replication in Ae. aegypti mosquito bodies, leading to a defect in viral dissemination to the salivary glands. Furthermore, CCR1 was shown not to regulate viral translation, RNA synthesis or virion retention in BHK or C6/36 cells but rather to modulate viral assembly, as mutations resulted in the release of non-infectious virions. Whereas most of the identified RNA elements in DENV have been shown to regulate RNA synthesis or viral translation, our data has implicated CCR1 as an assembly signal whose function is more important for the mosquito vector rather than the mammalian host. Understanding the role of cis-acting regulatory RNA elements in the DENV coding region will provide insight into viral replication strategies and may uncover novel anti-viral drug targets.