Borrelia burgdorferi, the Lyme disease pathogen, dramatically alters its protein profile when it is transmitted between ticks and mammals. Several differentially expressed proteins have been shown to be critical for the enzootic cycle of B. burgdorferi. In this study, we demonstrated that expression of the surface lipoprotein-encoding gene bba07 is induced by an elevated temperature and a reduced pH during in vitro cultivation, as well as during nymphal tick feeding. Expression of bba07 is regulated by the Rrp2-RpoN-RpoS pathway, a central regulatory network that is activated during nymphal feeding. By generating a bba07 mutant of an infectious strain of B. burgdorferi, we demonstrated that although BBA07-deficient spirochetes were capable of infecting mice via needle inoculation and surviving in ticks, they were defective in infection of mammals via tick transmission. Complementation of the bba07 mutant with a wild-type copy of bba07 partially restored the transmission defect of the bba07 mutant. Based on these findings, we concluded that the surface lipoprotein BBA07 is produced during tick feeding and facilitates optimal transmission of B. burgdorferi from the tick vector to a mammalian host.