Genital inflammation is associated with increased HIV acquisition risk. Induction of an inflammatory response can occur through the recognition of pathogenic or commensal microbes by Toll-like receptors (TLRs) on various immune cells. We used a in vitro peripheral blood mononuclear cell (PBMC) system to understand the contribution of TLR stimulation in inducing inflammation and the activation of target T cells, and its effect on HIV susceptibility. PBMCs were stimulated with TLR agonists LPS (TLR4), R848 (TLR7/8), and Pam3CSK4 (TLR1/2), and then infected with HIV NL4-3 AD8. Multiplexed ELISA was used to measure 28 cytokines in cell culture supernatants. Flow cytometry was used to measure the activation state (CD38 and HLA-DR), and CCR5 expression on CD4+ and CD8+ T cells. Although TLR agonists induced higher cytokine and chemokine secretion, they did not significantly activate CD4+ and CD8+ T cells and showed decreased CCR5 expression relative to the unstimulated control. Despite several classes of inflammatory cytokines and chemokines being upregulated by TLR agonists, CD4+ T cells were significantly less infectable by HIV after TLR4-stimulation than the unstimulated control. These data demonstrate that the inflammatory effects that occur in the presence TLR agonist stimulations do not necessarily translate to the activation of T cells. Most importantly, the finding that TLR4-stimulation reduces rather than increases susceptibility of CD4+ T cells to HIV infection in this in vitro system strongly suggests that the increased chemokine and possible antiviral factor expression induced by these TLR agonists play a powerful although complex role in determining HIV infection risk.Keywords: HIV; Toll-like receptors; cytokines; immune activation; inflammation; innate antiviral immunity.