Various plants have been studied for their therapeutic potential to treat virusinduced diseases. This study aimed to investigate in vitro anti-coxsackievirus B3 (CVB3) effect of saucernetin-8 isolated from Schisandra chinensis and mode of its action. The action of saucernetin-8 on CVB3 proliferation, the effect of saucernetin-8 on VP3 protein, mitochondrial reactive oxygen species (mtROS) and expression levels of STING/p-STING/IRF3/p-IRF3 were investigated by RT-qPCR, immunofluorescence and Western blot. Saucernetin-8 exhibited over 60% inhibition of CVB3-induced cytotoxicity at a concentration of 0.4 μg/mL. Saucernetin-8 completely suppressed viral genome replication at a concentration of 10 μg/mL. Furthermore, saucernetin-8 inhibited the expression of VP3, the capsid protein of CVB3, by approximately 50% at a concentration of 10 μg/mL. Immunofluorescence assay showed that saucernetin-8 inhibited VP3 expression by approximately 50%. Saucernetin-8 increased mtROS levels in CVB3-infected Vero cells. The STING/IRF3 pathway is activated in cells treated with saucernetin-8. Therefore, saucernetin-8 is a candidate for development of antivirals against CVB3.