Hydroxychloroquine (HCQ) is an antimalarial drug also used in treating autoimmune diseases. It is also an autophagosome-lysosome fusion inhibitor broadly used in autophagy pathway research. HCQ is a weak basic drug that accumulates in the acidic environment of cellular organelles to inhibit replication of different viruses by interfering with endosome/lysosome trafficking or viral protein maturation during virions maturation. We demonstrated that HCQ could inhibit four serotypes of dengue viruses (DENV1-4) infection in cells by immunofluorescence assays. RT-qPCR analysis of HCQ-treated cells showed induced expression of antiviral genes and cytokines such as interferon beta (IFNβ), IFN-induced protein with tetratricopeptide repeats 3, C-X-C motif chemokine 10, melanoma differentiation-associated protein 5, mitochondrial antiviral signaling protein (MAVS) and tumor necrosis factor receptor-associated factor 3. The expression of inflammatory cytokines such as interleukin 6 (IL-6), IL-12 p19, IL-12 p40 and tumor necrosis factor α were also induced by HCQ. Mechanistic study suggested that HCQ activated the innate immune signaling pathways of IFNβ, AP-1 and NFκB via phosphorylation of IFN regulatory factor 3 and c-Jun and by increasing NFκB p65 subunit nuclear translocation. Blocking type I IFN by antibody targeting IFN receptor or by inhibiting MAVS, TBK1 and IKKε signaling reduced the efficiency of HCQ against DENV-2 infection. These results reveal an emerging role of HCQ activating the host innate immunity against virus infection.