Human Cathelicidin antimicrobial peptide LL-37 is known to have antiviral activity against many viruses. In the present study, we investigated the in-vitro effect of LL-37 on dengue virus type 2 (DENV-2) infection and replication in Vero E6 cells. To study the effect of pretreatment of virus or cells with LL-37, the virus was pretreated with different concentrations of LL-37 (2.5 μM–15 μM) or scrambled (Scr) LL-37(5 μM–15 μM) and used for infection or the cells were first treated with LL-37 and infected. To study the effect of LL-37 post infection (PI), the cells were infected first followed by addition of LL-37 to the culture medium 24 h after infection. In all conditions, after the incubation, the culture supernatant was assessed for viral RNA copy number by real time RT-PCR, infectious virus particles by focus forming unit assay (FFU) and non structural protein 1 (NS1) antigen levels by ELISA. Percentage of infection was assessed using immunoflourescence assay (IFA). The results revealed that pretreatment of virus with 10–15 μM LL-37 significantly reduced its infectivity as compared to virus control (P < 0.0001). Moreover, pretreatment of virus with 10–15 μM LL-37 significantly reduced the levels of viral genomic RNA and NS1 antigen (P < 0.0001). Treatment of virus with 10–15 μM LL-37 resulted in two to three log reduction of mean log10 FFU/ml as compared to virus control (P < 0.0001). Treatment of the virus with scrambled LL-37 had no effect on percentage of infection and viral load as compared to virus control cultures (P > 0.05). Pretreatment of cells before infection or addition of LL-37 to the culture 24 h PI had no effect on viral load. Molecular docking studies revealed possible binding of LL-37 to both the units of DENV envelope (E) protein dimer. Together, the in-vitro experiments and in-silico analyses suggest that LL-37 inhibits DENV-2 at the stage of entry into the cells by binding to the E protein. The results might have implications for prophylaxis against DENV infections and need further in-vivo studies.