Diospyrin, plant-derived bisnaphthoquinonoid, is known to have anticancer activity. However, pharmacological activity of diospyrin on viral infection is not well known. We investigated effects of diospyrin on macrophages induced by polyinosinic-polycytidylic acid (poly I:C), a mimic of double-stranded viral RNA. Various cytokines, intracellular calcium, nitric oxide (NO), phosphorylated p38 MAPK, and phosphorylated ERK1/2 as well as mRNA expressions of transcription factors were evaluated. Diospyrin significantly reduced NO production, granulocyte-macrophage colony-stimulating factor production, and intracellular calcium release in poly I:C-induced RAW 264.7. The phosphorylation of p38 MAPK and ERK1/2 was also significantly suppressed. Additionally, diospyrin inhibited mRNA levels of nitric oxide synthase 2, C/EBP homologous protein (CHOP), calcium/calmodulin dependent protein kinase II alpha, signal transducers and activators of transcription 1 (STAT1), STAT3, STAT4, Janus kinase 2, first apoptosis signal receptor, c-Jun, and c-Fos in poly I:C-induced RAW 264.7. Taken together, this study represents that diospyrin might have the inhibitory activity against viral inflammation such as excessive production of inflammatory mediators in poly I:C-induced RAW 264.7 via ER stress-induced calcium-CHOP pathway.