dsRNA is frequently associated with viral replication. Here, we compared the costimulatory effect of the synthetic analog of dsRNA, poly(I:C), and the agonistic anti-CD28 mAb on anti-CD3 mAb-activated, freshly isolated human CD4 T cells. We tested the hyphothesis that poly(I:C) and anti-CD28 mAb costimulation differ in their effect on the CD4 T cell immune response. Our study shows that costimulation of CD4 T cells by poly(I:C) enhanced CD3-induced production of IP-10, MIP1-α/β, RANTES, and granzyme B involved in antiviral activity more than anti-CD28 mAb. poly(I:C) stimulation, on its own, activated the transcription of IRF7 in human CD4 T cells. Combined CD3 and poly(I:C) stimulation significantly enhanced the transcription of IRF7 and additionally, NF-κBp65 phosphorylation, which might be involved in the induction of antiviral chemokines and the enhanced cytotoxic activity of poly(I:C)-treated CD4 T cells. In comparison with poly(I:C), anti-CD28 mAb as a costimulus induced a stronger proinflammatory response, as indicated by enhanced TNF-α secretion. poly(I:C) had a costimulatory effect on Akt phosphorylation, whereas anti-CD28 mAb only slightly enhanced Akt phosphorylation. In contrast to poly(I:C), anti-CD28 mAb was essential for proliferation of anti-CD3-stimulated CD4 T cells; however, poly(I:C) further increased the anti-CD28/anti-CD3-mediated proliferation. These results indicate that poly(I:C)- and anti-CD28 mAb-induced signaling differ in their costimulatory effect on the CD3-driven, antiviral chemokine release and proinflammatory cytokine secretion in freshly isolated human CD4 T cells.