Borrelia burgdorferi preferentially induces selected genes in mice or ticks, and studies suggest that ospD is down-regulated in response to host-specific signals. We now directly show that ospD expression is generally elevated within Ixodes scapularis compared with mice. We then assessed the importance of OspD throughout the spirochete life cycle by generating OspD-deficient B. burgdorferi and examining the mutant in the murine model of tick-transmitted Lyme borreliosis. The lack of OspD did not influence B. burgdorferi infectivity in mice or the acquisition of spirochetes by I. scapularis. OspD adhered to tick gut extracts in vitro, and the OspD-deficient B. burgdorferi strain had a threefold decrease in colonization of the tick gut in vivo. This decrease, however, did not alter subsequent spirochete transmission during a second blood meal. These data suggest that B. burgdorferi can compensate for the lack of OspD in both ticks and mice and that OspD may have a nonessential, secondary, role in B. burgdorferi persistence within I. scapularis.