Chitobiose is the dimer subunit of chitin, a component of tick cuticle and peritrophic matrix, which is not found in mammals. The Borrelia burgdorferi chbC gene is required for the use of chitobiose as a source of the essential nutrient N-acetyl glucosamine during growth in vitro. In order to investigate the role of chitobiose transport in the infectious cycle, we constructed isogenic chbC mutant and wild-type strains in an infectious B. burgdorferi background and confirmed that the mutants were defective in chitobiose utilization. The defect in the mutants was shown to be in chitobiose transport, consistent with the predicted function of the ChbC protein as the membrane component of a phosphotransferase transporter for chitobiose. We then tested whether this locus is also required for any stage of the experimental mouse-tick infectious cycle. We found that both wild-type and mutant bacteria successfully infect both mice and ticks and are transmitted between the two hosts. These results demonstrate that B. burgdorferi growth in vivo is independent of chitobiose transport, even in an environmental niche in which the sugar is likely to be present.