Indoleamine 2,3-dioxygenase (IDO) is an enzyme that degrades an essential amino acid, tryptophan, and plays a role in inhibiting the proliferation of T cells and intracellular pathogens. Inhibiting IDO in mice leads to fetal rejection, suggesting its significance in establishing pregnancy. Toll-like receptor 3 (TLR-3) is a key component of the innate immune system that recognizes viral double-stranded RNA and triggers immune reactions by producing type I interferon. Using a human trophoblast cell culture system, we studied the effect of TLR-3 ligation on IDO expression and function by treating trophoblasts with polyinosinic-polycytidylic acid [poly(I:C)] (a synthetic double stranded RNA, which mimics viral RNA). Real-time PCR and Western blot analysis revealed that IDO mRNA and protein expression was significantly induced by poly(I:C). The activity of IDO was also increased by poly(I:C) given that the L-kynurenine concentrations were elevated in conditioned media. Conditioned media from poly(I:C)-treated trophoblasts were found to inhibit the proliferation of human T cells significantly. Poly(I:C) was also shown to induce interferon (IFN)-β mRNA expression in trophoblasts. Recombinant human IFN-β increased IDO mRNA expression in trophoblasts more rapidly than poly(I:C). Pretreating with neutralizing antibody against IFN-β significantly suppressed IDO induction by poly(I:C). Collectively we have demonstrated that ligation of TLR-3 by poly(I:C) induces IDO expression in human first-trimester trophoblasts via an IFN-β-dependent pathway. These findings suggest that upon viral infection, trophoblasts induce IDO and in turn contribute to antimicrobial activity and maintenance of fetomaternal tolerance.