Flaviviruses including Zika virus, Dengue virus, Japanese Encephalitis virus and Yellow Fever virus cause heavy burdens to public health around the world. No specific antiviral drug was available in clinic against these flavivirus infections. Heat-shock protein 70 (HSP70) has recently been proven to be a promising antiviral target against Zika virus and Dengue virus. Here, we report that, Apoptozole, a small molecule inhibitor of ATPase activity of HSP70 has broad-spectrum anti-flavivirus potential. The mode of action analysis revealed that Apoptozole acted at post-entry step. Transcriptome analysis revealed that genes related to cholesterol metabolism, fatty acid synthesis and innate immunity were differentially expressed after treatment of Apoptozole. In vivo data suggested Apoptozole exerted protection effects against ZIKV infection in mouse model by enhancing innate immune response, which suggested a novel anti-ZIKV mechanism of HSP70 inhibitors. Key words: Flavivirus, Antiviral, Apoptozole, Zika virus, Innate immunity.