Dengue virus (DENV) is a type of arthropod-borne Flavivirus, which leads to a series of serious diseases like dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DENV has a devastating health and economic impact worldwide. However, there are no suitable drugs to combat the virus. Here we reported that HSPA13, also known as stress chaperone (STCH), is a member of the HSP70 family and is a key regulator of type I interferon (IFN-I) and pro-inflammatory responses during DENV infection. HSPA13 expression was increased in macrophages infected with DENV or other Flaviviruses like Zika virus (ZIKV), Yellow fever virus (YFV) and Japanese encephalitis virus (JEV). Further, HSPA13 suppressed the replication of DENV and other Flaviviruses (ZIKV, JEV, YFV), which exhibited broad-spectrum antiviral effects. On the one hand, HSPA13 promoted production of IFN-β and interferon-stimulated genes (ISGs, such as ISG15, OAS and IFIT3) by interacting with RIG-I and up-regulating RIG-I expression during DENV infection. On the other hand, HSPA13 enhanced NLRP3 inflammasome activation and IL-1β secretion by interacting with ASC in DENV infection. We identified HSPA13 as a potential anti-DENV target. Our results provide clues for the development of antiviral drugs against DENV based on HSPA13 and reveal novel drug target against Flaviviruses.Copyright © 2023. Published by Elsevier B.V.