Borrelia burgdorferi lipoprotein Lp6.6 is a differentially produced spirochete antigen. An assessment of lp6.6 expression covering representative stages of the infectious cycle of spirochetes demonstrates that the gene is solely expressed during pathogen persistence in ticks. Deletion of lp6.6 in infectious B. burgdorferi did not influence in vitro growth, or its ability to persist and induce inflammation in mice, migrate to larval or nymphal ticks or survive through the larval-nymphal molt. However, Lp6.6-deficient spirochetes displayed significant impairment in their ability to transmit from infected ticks to naïve mice, which was restored upon genetic complementation of the mutant with a wild-type copy of lp6.6, establishing that Lp6.6 plays a role in pathogen transmission from ticks to mammals. Lp6.6 is a subsurface, yet highly abundant, outer membrane antigen. Two-dimensional blue native/SDS-PAGE coupled with liquid chromatography-mass spectrometry (LC-MS/MS) analysis and protein cross-linking studies independently shows that Lp6.6 exists in multiple protein complexes in the outer membrane. We speculate that the function of Lp6.6 is connected to the physiological processes of these membrane complexes. Further characterization of differentially produced membrane antigens and associated protein complexes will likely aid in our understanding of the molecular details of B. burgdorferi persistence and transmission through a complex enzootic cycle.