Infection with the mosquito transmitted dengue virus (DENV) remains a significant worldwide public health problem. While the majority of infections are asymptomatic, infection can result in a range of symptoms. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through repression or degradation of mRNAs. To understand the contribution of miRNAs to DENV 2 replication, we screened a number of candidate miRNAs for variations in expression levels during DENV 2 infection of HepG2 (liver) cells. Seven miRNAs were identified as differentially expressed, and one, miR-21, was differentially expressed at all time points examined. Interestingly, miR-21 was also differentially regulated in DENV 2 infection under conditions of antibody dependent enhancement of infection, and in direct Zika virus infection, but not in DENV 4 infection. The role of miR-21 during DENV infection was further examined by treating HepG2 cells with an anti-miR-21 (AMO-21) before DENV infection. The results showed a significant reduction in DENV 2 production, clearly suggesting that miR-21 plays a key role in DENV 2 replication. To further confirm the role of miR-21 in DENV infection, a peptide nucleic acid-21 (PNA-21) construct with a nucleotide sequence complementary to AMO-21, was co-administered with AMO-21 as an AMO-21/PNA-21 complex followed by DENV 2 infection. The results showed that AMO-21 significantly reduced DENV 2 titer, PNA-21 significantly increased DENV 2 titer and the combined AMO-21/PNA-21 showed no difference from non-treated infection controls. Taken together, the results show that miR-21 promotes DENV 2 replication, and this mechanism could serve as a possible therapeutic intervention point.