It has been proposed that the systemic immune activation state seen in HIV-1-infected patients is caused by circulating microbial products such as lipopolysaccharide (LPS). Given that macrophages play a key role in HIV-1 pathogenesis, we investigated the LPS-mediated effect on HIV-1 replication in cells of the myeloid lineage. We demonstrate that LPS promotes virus gene expression in a monocytic cell line while it diminishes virus production in primary human monocyte-derived macrophages (MDM). The incapacity of LPS to drive HIV-1 production in MDM was not due to its inability to activate the ubiquitous transcription factor NF-κB even in virus-infected cells. Neutralization of type I interferons (IFN) with B18R, a soluble vaccinia virus-coded type I IFN receptor, significantly but not totally diminished the antiviral activity of LPS. Therefore, inhibition of HIV-1 replication in MDM treated with microbial-derived LPS resulted from the induction of type I interferons and a yet to be defined soluble factor.