Our study presents a first investigation of the effect of the adjuvant PIKA on dengue virus (DENV) replication. PIKA pretreatment decreased the levels of DENV serotype 2 (DENV2) mRNA, protein and viral particles in the hepatoma cell line HepG2. Treatment with PIKA simultaneously with DENV2 infection, but not after infection, resulted in a protective effect. Significant induction of type I and type III interferons (IFNs), as well as interferon-stimulated genes was detected in PIKA-pretreated cells. Neutralization of IFN-β partially restored the replication levels of DENV2 in PIKA-pretreated cells, suggesting that IFN-β is one of the mediators involved in the antiviral action of PIKA. Additionally, blockade of TBK-1 signaling largely restored the IFN induction and viral suppression effects mediated by PIKA, further illustrating that PIKA plays its anti-DENV role by promoting innate immunity. These findings suggest that PIKA is an attractive agent to be used in the prevention of DENV diseases.