<h3>Introduction</h3> Syphilis is a multi-stage, sexually transmitted disease caused by the spirochete <i>Treponema pallidum</i> (<i>Tp</i>). Clinical manifestations result from the treponeme's ability to elicit a robust immune response while at the same time evading host defenses. Syphilitic lesions are comprised of a rich cellular infiltrate, which includes IFN-gamma (IFNg) producing T cells, NK cells and activated macrophages. We previously, we demonstrated that human syphilitic serum (HSS) promotes efficient uptake of <i>Tp</i> by human monocytes and that opsonophagocytosis of <i>Tp</i> markedly enhances cytokine production. The purpose of this study is to establish a potential role for macrophages and opsonic Ab in clearance of <i>Tp</i> and generation of tissue-based inflammation during human syphilis. <h3>Methods</h3> We used monocyte-derived macrophages to develop an <i>ex vivo</i> model for studying spirochete-macrophage interactions. We used macrophage-colony stimulating factor and IFNg for macrophage maturation and evaluated the immunophenotypic modulations by flow cytometry. We assessed <i>Tp</i> uptake, in the presence or absence of HSS by confocal microscopy. We also determined the cellular responses initiated by opsonophagocytosis of <i>Tp</i> using targeted transcriptional array analysis and cytokine bead array. <h3>Results</h3> IFNg polarisation of macrophages led to an increase in Fcg receptors (FcgRs) expression, phagocytosis of HSS opsonized <i>Tp</i> and cytokine production. Blockade of CD64 significantly diminished spirochetal uptake and pro-inflammatory cytokine secretion by the macrophages. <h3>Conclusion</h3> Our <i>ex vivo</i> studies provide a potential role for macrophages in clearance of <i>Tp</i> during human syphilis. These data are the first to demonstrate that CD64 in the primary FcR involved in opsonophagocytosis of <i>Tp</i> and IFNg plays a critical role in the macrophages responsiveness following uptake of the spirochete. Moreover, our study results also provide an <i>ex vivo</i> surrogate system for use in future syphilis vaccine studies.