Galectin-9 is a member of the galectin family, which induces various biological reactions such as chemotaxis of eosinophils and apoptosis of T cells. We previously reported that polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA (dsRNA), induces the expression of galectin-9 in human umbilical vein endothelial cells (HUVECs). In the present study, we addressed the possible involvement of two potential receptors for dsRNA, Toll-like receptor (TLR) 3 and retinoic acid-inducible gene-I (RIG-I), in the expression of galectin-9 in HUVECs. Poly IC-induced galectin-9 expression was almost completely suppressed by RNA interference (RNAi) against TLR3, but not against RIG-I. LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited the induction of galectin-9 by poly IC. RNAi against interferon regulatory factor 3 (IRF3) also inhibited poly IC-induced galectin-9 expression. We conclude that TLR3, PI3K, and IRF3 are involved in the poly IC-induced galectin-9 expression in HUVECs.