Author Summary: Syphilis remains a major cause of morbidity and mortality worldwide. The bacterium causing syphilis, Treponema pallidum ssp. pallidum , has evolved into a highly distinctive organism that is only able survive (and be propagated) in mammals. In humans it can evade the immune system for decades with devastating consequences. Much remains to be learned about how it accomplishes this. Only a minority of its predicted proteins have been detected experimentally thus far. We aimed to more comprehensively characterize the proteins of this organism. Since it cannot be cultured in vitro , we cultured T . pallidum in rabbits and analyzed extracted proteins using different mass spectrometry methods, a manner of detecting proteins with high accuracy. In total, we detected more than half of the predicted number of proteins that could be expressed by this bacterium (N = 557). For approximately half of the proteins, we succeeded in characterizing their predicted cellular location using an array of bioinformatic tools and catalogued their function. This is the most comprehensive analysis of the T . pallidum proteome to date. This study lays the groundwork for other protein investigations of this unique organism.