Background: Activated platelets secrete platelet factor 4 (PF4), which contributes to viral pathogenesis. Recently, we reported the proviral role of PF4 in replication of closely related flaviviruses, Japanese encephalitis virus (JEV) and dengue virus (DENV). Objectives: This study aimed to investigate the detailed mechanism of PF4-mediated virus replication. Methods: PF4-/- or wild-type (WT) mice were infected with JEV, and host defense mechanisms, including autophagic/interferon (IFN) responses, were assessed. WT mice were pretreated with the CXCR3 antagonist AMG487 that inhibits PF4:CXCR3 pathway. This pathway was tested in PF4-/- monocytes infected with DENV or in monocytes isolated from patients with DENV infection. Results: PF4-/- mice infected with JEV showed reduced viral load and improved brain inflammation and survival. PF4-/- mice synthesized more IFN-α/β with higher expression of phosphorylated IRF3 in the brain. PF4 treatment decreased IRF-3/7/9 and IFN-α/β expression and suppressed autophagic LC3-II flux and lysosomal degradation of viral proteins in JEV-infected cells. PF4 increased the expression of P-mTOR, P-p38, and P-ULK1Ser757 and decreased expression of LC3-II. Decreased autophagosome-lysosome fusion in turn promoted DENV2 replication. The above processes were reversed by AMG487. Uninfected PF4-/- monocytes showed elevated LC3-II and autophagosome-lysosome fusion. Microglia of JEV-infected PF4-/- mice exhibited elevated LC3-II inversely related to viral load. Similarly, monocytes from PF4-/- mice showed reduced infection by DENV2. In patients with DENV infection, higher plasma PF4 and viral load were inversely correlated with LC3-II, LAMP-1, and lysosomal degradation of DENV-NS1 in monocytes during the febrile phase. Conclusion: These studies suggest that PF4 deficiency or inhibition of the PF4:CXCR3 pathway prevents JEV and DENV infection. The studies also highlight the PF4:CXCR3 axis as a potential target to develop treatment regimens against flaviviruses.Keywords: CXCR3; DENV; JEV; autophagy; infection; interferon; mice; patients; platelet factor 4.