Syphilis is a sexually transmitted infection caused by the highly invasive and immunoevasive spirochetal pathogen subsp. (). Untreated syphilis can lead to infection of multiple organ systems, including the central nervous system. The alarming increase in syphilis cases globally underscores the importance of developing novel strategies to understand the complexities of syphilis pathogenesis. In this study, we took advantage of recent advances in cultivation and genetic manipulation of syphilis spirochetes to engineer a strain that constitutively expresses green fluorescent protein (GFP). GFP grew identically to the Nichols parent strain and exhibited wild-type infectivity in the rabbit model. We then used the GFP strain to visualize interactions with host cells during co-cultivation , within infected rabbit testes, and following opsonophagocytosis by murine bone marrow-derived macrophages. Development of fluorescent strain also enabled us to develop a flow cytometric-based assay to assess antibody-mediated damage to the spirochete's fragile outer membrane (OM), demonstrating dose-dependent growth inhibition and OM disruption . Notably, we observed greater OM disruption of GFP with sera from immune rabbits infected with the Nichols strain compared to sera generated against the genetically distinct SS14 strain. These latter findings highlight the importance of OM protein-specific antibody responses for clearance of during syphilitic infection. The availability of fluorescent strains paves the way for future studies investigating spirochete-host interactions as well as functional characterization of antibodies directed treponemal OM proteins, the presumptive targets for protective immunity.