The recognition of pathogen-associated molecular patterns (PAMPs) by the innate immune system is a crucial step in inducing effective immune responses. Double-stranded RNA [mimicked by polyinosinic-polycytidylic acid (poly(I:C)], synthesized by various types of viruses, represents one important member of these immunostimulatory microbial components. Here we report that poly(I:C) has potent γδ T-cell costimulatory capacity. Within peripheral blood mononuclear cells, poly(I:C)-stimulated γδ T cells expressed increased levels of CD69 and exhibited significantly enhanced antigen-mediated proliferation in response to isopentenylpyrophosphate (IPP). Among several recombinant cytokines tested, type I interferons (IFN-α, IFN-β) and interleukin-15 (IL-15) showed a similar activation pattern of γδ T cells. γδ T-cell clones and purified γδ T cells did not respond to poly(I:C), indicating indirect effects of this compound. Depletion of CD11c+ dendritic cells (DC), which express Toll-like receptor 3 (TLR3), known to recognize poly(I:C), abrogated poly(I:C)-mediated stimulation of γδ T cells. In addition, the supernatant of poly(I:C)-treated CD11c+ DC was able to mimic the stimulatory effects of poly(I:C) on γδ T cells. Experiments with neutralizing antibodies indicated that type I IFNs, but not IL-15, contributed to the poly(I:C)-mediated activation of γδ T cells. In conclusion, γδ T-cell activation by immunostimulatory double-stranded RNA, such as poly(I:C), is indirectly mediated via type I IFNs derived from TLR3-expressing CD11c+ DCs. These results suggest that upon confrontation with certain viruses, γδ T cells can be rapidly activated by type I interferons and may contribute to effective antiviral responses.