The cell biology of viral infections is the focus of this research, in which the role of the cytoskeleton in dengue virus (DENV) replication in cell cultures was evaluated by means of Nocodazole and Cytochalasin D treatments before and after of DENV infection. The potential contribution of cytoskeleton elements with/without the treatment of depolymerizing agents was evidenced and quantified by the subcellular distribution of viral proteins, virions produced, and viral protein quantification. The cytoskeleton is involved in DENV replication because treatments with actin microfilaments and microtubule depolymerizing agents in non-cytotoxic concentrations, affected DENV2 replication in Vero cells and decreased both the viral protein expression and infectious virion production, when compared with non-treated cells. The actin and microtubules are partly involved in DENV2 replication, since the treatment does not completely blocked viral replication, suggesting that these components are necessary but not sufficient alone for DENV2 replication in Vero cells. The structural and functional role of actin and the microtubules in replication are postulated here, opening new perspectives for understanding the architecture of the replicative complex and viral morphogenesis processes, due to the role of the cytoskeleton in the organization, recruitment, and function of the cellular elements necessary for the assembly of viral factories.