Human papilloma virus (HPV)-like particles (VLPs) have been used as a vaccine to prevent HPV infection. Recent studies demonstrate that VLPs bind to dendritic cells and induce the expression of antiviral cytokines such as interferon-alpha (IFN-alpha), interleukin-10 (IL-10) and IFN-gamma. In the present study, we evaluated the effect of VLPs on HIV-1 replication in peripheral blood mononuclear cells (PBMCs), CD4+ T cells, and macrophages. Here, we show that VLPs suppress the replication of both X4 and R5 HIV-1 without affecting the expression of CD4, CXCR4, and CCR5. Soluble factor(s) released by PBMCs and macrophages on VLPs treatment inhibited HIV-1 replication. To determine the inhibitory factors, DNA microarray analysis was performed using VLP-treated PBMCs and macrophages. VLPs induced the genes associated with IFN induction, immune responses, and antiviral responses, among with the recently described cytokine IL-27. Subsequently, IL-27 was found to be a potent inhibitor of HIV-1 replication in PBMCs, CD4+ T cells, and macrophages. Taken together, our studies identify a novel role of IL-27 in restricting HIV-1 replication and suggest that further examination of the inhibitory property of IL-27 may pave the way for a novel therapy for HIV-1 infection.